JP4277956B2 - Compositions and methods for increasing cholesterol efflux and raising HDL using the ATP binding cassette transporter protein ABC1 - Google Patents

Description

  This application is filed in US Provisional Application No. 60 / 140,264, filed June 18, 1999; US Provisional Application No. 60 / 153,872 filed September 14, 1999; and November 19, 1999. Claims priority to US Provisional Application No. 60 / 166,573, filed on the same day.

(Technical field of the invention)
The present invention relates to novel ABC1 polypeptides and nucleic acid molecules encoding the same. The invention also relates to a recombinant vector, a host cell, and a composition comprising an ABC1 polynucleotide, and a method for producing an ABC1 polypeptide. The present invention also relates to an antibody that specifically binds to ABC1 polypeptide. In addition, the present invention relates to methods for increasing cholesterol efflux and methods for increasing ABC1 expression and activity. The invention further relates to methods for identifying compounds that modulate ABC1 expression and methods for detecting comparative levels of ABC1 polypeptides and polynucleotides in mammalian subjects. The invention also provides kits and compositions suitable for screening compounds to measure the ABC1 expression modulating activity of the compounds, as well as determining whether a compound modulates ABC1-dependent cholesterol efflux. Suitable kits and compositions are provided.

  Circulating lipids in human plasma or lymph consist of cholesterol, cholesteryl esters, triglycerides and phospholipids. These lipids are transported in large molecular complexes called lipoproteins, which consist of a cholesteryl ester and / or triglyceride core, a phospholipid and free cholesterol envelope, an apolipoprotein (Scriver et al., Edit., The Metabolic. and Molecular Basis of Inherited Disease, 7th edition, p. 1841-1851 (McGraw-Hill, New York 1995)). Apolipoproteins are involved in the assembly and secretion of lipoproteins and the activation of lipoprotein modifying enzymes such as lecithin cholesterol acyltransferase (LCAT). In addition, apolipoproteins provide structural integrity, which is a ligand and membrane docking protein for large spectrum receptors. Plasma lipoproteins are divided into five types according to size: kilomicrons (largest size and lowest density), very low density lipoprotein (VLDL), medium density lipoprotein (IDL), and low density lipoprotein (LDL). ) And high density lipoprotein (HDL).

  Kilomicron, VLDL, IDL and LDL transport exogenous and endogenous cholesterol and triacylglycerol to peripheral sites, where lipids play a role in various metabolic pathways and serve as major components of cell membranes. Kilomicrons are assembled in the intestinal mucosa as a means of transporting diet cholesterol and triacylglycerol to various tissues. VLDL is formed in the liver and transports endogenous cholesterol and triacylglycerol synthesized by the liver to extrahepatic tissues such as muscle and desired tissues. VLDL contains 10-15% synthetic serum cholesterol and most triglycerides. In the circulation, VLDL is converted to LDL through the action of lipoprotein lipase. LDL is the primary plasma carrier of cholesterol for delivery to all tissues and typically contains 60-70% total fasted serum cholesterol.

  In contrast, HDL is involved in “reverse cholesterol transport”, a pathway in which excess cholesterol is transported back from the peripheral site to the liver where it is secreted in the form of bile salts (Glomset, JA, J. Lipid Res., 9, 155-167 (1968)). Freshly generated HDL is synthesized de novo in the liver and small intestine as disk-shaped particles rich in proteins lacking cholesterol and cholesterol esters. In fact, the main function of HDL acts as a circulating store of apolipoproteins, mainly apoC-I, apoC-II and apoE. Freshly generated or protein rich HDL is converted to spherical lipoprotein particles through the accumulation of cholesteryl esters obtained from cell sources. HDL usually contains 20-30% total fasted serum cholesterol.

  According to current theory, the reverse efflux of cellular cholesterol into HDL is mediated by two mechanisms: an aqueous diffusion pathway and an apolipoprotein-mediated pathway. The relative importance of these distinguishable mechanisms depends on cell type and metabolic status (Oram et al., J. Lipid Res., 37: 2743-2491 (1996); Rothblat et al., J. Lipid Res., 40 : 781-796 (1999); Stein et al., Atherosclerosis, 144: 285-301 (1999)). In many cells, the aqueous diffusion pathway is the primary pathway through which cholesterol efflux occurs (Jhonson et al., Diochim. Biophys. Acta, 1085: 293-298 (1991)). This pathway involves a bidirectional exchange of cholesterol between the cell membrane and lipoprotein receptors in the extracellular space through the process of passive transport (Remaley et al., Arterioscler. Thromb. Vasc. Biol., 17: 1833- Rothblat et al., J. Lip.Res., 40: 781-796 (1999) The exchange can occur primarily as surface microdomains known as vesicles (Fielding et al., Biochemistry, 34). : 14292 91995)). Net efflux can be driven by the conversion of cholesterol cholesteryl esters in the extracellular compartment by the action of LCAT.

  Alternatively, in macrophages and fibroblasts, cholesterol and phospholipid efflux are mediated primarily through apolipoproteins such as ApoA-I, ApoA-II and ApoE (Remaley, supra (1997); Francis, et al., J. Clin. Invest., 96: 78-87 (1995); Vega et al., J.Intern.Med., 226: 5-15 (1989); Sakar et al., Biochim.Biophys. Acta, 1438: 85-98. Hara et al., J. Biol. Chem., 266: 3080-3086 (1991); Fileding et al., J. Lipid Res., 38, 1503-1521 (1997); Oram et al., J. Lipid Res. ., 37, 2 743-2491 (1996)). The process of apolipoprotein-mediated lipid efflux is particularly prevalent in macrophages and other scavenger cells when it is loaded with cholesterol and / or when growth is inhibited. Apolipoprotein-mediated efflux is an active transport process that requires direct interaction between apolipoprotein and the cell surface, lipidation of apolipoprotein, and subsequent dissociation of lipid-apolipoprotein particles from the cell ( Oram, supra (1996); Mendez, AJ, J. Lipid Res., 38, 1807-1821 (1997); Remaly, supra (1998); Mendez, AJ, J. Lipid Res., 37 2510-2524 (1996)). Once removed from the cell, the cholesterol is rich and HDL particles are transported to the liver and removed from the body as described.

  Abnormal lipoprotein function and / or metabolism resulting from a genetic defect or as a secondary effect of another disorder can have serious biological consequences. In addition to diet effects, disorders such as diabetes, hypothyroidism and liver disease result in elevated plasma levels of LDL-cholesterol and triglyceryl. Elevated levels of LDL-cholesterol and triglycerides have been identified as major risk factors associated with the incidence of coronary heart disease, a leading cause of death in the United States and other industrialized countries (Hokason et al., J Cardiovasc.Risk., 3: 213-219 (1996); The Expert Panel, JAMA, 269: 3015-3023 (1993)). Accumulation of excess LDL-cholesterol in the arterial wall can lead to the formation of atherosclerotic plaques, which play a major role in the development of heart disease. Plaque is believed to be formed when free radicals released from the arterial wall oxidize LDL. According to theory, the oxidized form of LDL triggers an inflammatory response, attracting circulating cells to sites that contribute to the formation of lipid plaques. Among these are other cells containing scavenger receptors that accumulate macrophages and cholesterol in an unregulated manner (Brown et al., Ann. Rev. Biochen., 52: 223-261 (1986)). The massive storage of internal cholesterol results in a conversion to a foam cell phenotype, which is thought to be a major contributor to the development of vascular lesions. As plaque is formed, the arterial wall contracts, reducing blood flow to the heart.

  Interestingly, however, an estimated 60% of heart attacks occur in people who do not have elevated blood levels of LDL-cholesterol. Of these, an estimated 45% is associated with HDL-cholesterol below average blood levels, indicating that low HDL-cholesterol levels are a significant risk factor for coronary heart disease. In fact, recent studies have shown that reduced HDL-cholesterol levels are the most common lipoprotein abnormality observed in patients with immature coronary artery disease (Genest J., Circulation, 85: 2025). -2033 (1992); Genest et al., Arterioscler. Thromb., 13: 1728-1737 (1993)). Although the basis for the inverse association between HDL-cholesterol and coronary heart disease is not well understood, the cardioprotective role of HDL is its related to the promotion of cholesterol efflux from macrophage foam cells in atherosclerotic lesions It was suggested that it may be derived from activity.

  One example of cardiovascular disease associated with low HDL is Tangier disease (TD), a rare genetic disorder characterized by little or complete absence of circulating HDL. In addition to almost zero plasma levels of HDL, patients with TD have massive deposition and accumulation of cholesteryl esters in several tissues including tonsils, lymph nodes, liver, spleen, thymus, intestine and hand and arm cells (Fredrickson) , DS, J. Clin. Invest., 43-228-236 (1964); Assmann et al., The Metabolic Basis of Inherited Disease, (McGraw-Hill, New York, 1995)). Although cellular mechanisms have not been previously identified, recent experiments have shown that cells from subjects with TD are deficient in the process of apolipoprotein-mediated removal of cholesterol and phospholipids (Remaley et al.,. Artiscler.Thromb.Vasc.Biol, 17,1813-1721 (1997); Francis et al., J.Clin, Invest., 96,78-87 (1995); Rogler et al., Arterioscler.Thromb. 15, 683-690 (1995)). These results stem from the inability of apoA-I, which has just developed severe HDL deficiency in TD patients, to acquire lipids. Since they do not mature into lipid-rich particles, freshly generated HDL in TD patients is rapidly catabolized and removed from the plasma, resulting in nearly zero levels of circulating HDL (Remaley supra (1997); Francis, supra (1995). Horowitz et al., J. Clin. Invest., 91, 1743-1752 (1993); Schaefer et al., J. Lip. Res., 22: 217-228 (1981)).

  Other disorders associated with premature atherosclerosis and high risk for coronary heart disease resulting from decreased HDL-cholesterol levels are hypoalphalipoproteinemia and familial HDL deficiency syndrome (FHA). People with these disorders often have normal LDL cholesterol and triglyceride levels. In addition, disorders such as diabetes, alcoholism, hypothyroidism, liver disease and elevated blood pressure result in decreased blood levels of HDL-cholesterol, although many of these disorders have increased LDL- Accompanying cholesterol and triglyceride levels.

  Current therapies for coronary heart disease have primarily focused on diet manipulation and / or drug treatments with the aim of reducing LDL-cholesterol blood levels by inhibiting LDL secretion or accelerated LDL turnover. . Derivatives of fibric acid such as clofibrate, gemfilrosyl and fenofibrate promote rapid VLDL turnover by activating lipoprotein lipase. Nicotinic acid reduces VLDL and LDL blood levels by inhibiting hepatic VLDL secretion. In addition, HMG-CoaA reductase inhibitors such as mebuinoline, mevastatin, pravastatin, simvastatin, flubutatin and lovastatin reduce plasma LDL levels by inhibiting the intracellular organization of cholesterol which causes increased cellular uptake of LDL . In addition, bile acid-binding resins such as colestillin, colestipol, and probucol reduce LDL-cholesterol levels by increasing catabolism of LDL-cholesterol in the liver.

  However, many of these therapies involve low efficiency and / or side effects that can hinder long-term use. For example, HMG-CoaA reductase inhibitors carry a considerable risk of toxicity. Because they inhibit the synthesis of mevalonate, which is necessary for the synthesis of other important isoprenoid compounds in addition to cholesterol. Gemfibrozil and nicotinic acid are also associated with severe adverse effects including kidney damage, muscle damage, muscle globinemia and unacceptable skin flushing and pruritus. In addition, the role of probucol in treating patients with coronary heart disease is uncertain. This is because its administration results in lower HDL-cholesterol levels as a side effect of lowering LDL-cholesterol.

  Furthermore, treatment of patients with isolated low HLDL-cholesterol levels provides a particularly challenging therapeutic challenge. For example, patients with Tangier disease exhibit a 4-6 fold increase in cardiovascular disease even though their LDL levels have already dropped by about 50%. Although there is some evidence that gemfibrozil and nicotinic acid simultaneously raise LDL levels, therapies aimed at lowering plasma LDL-cholesterol levels are generally associated with Tangier patients suffering from coronary heart disease as a result of reduced HDL levels. Not effective. Similarly, low alpha lipoproteinemia, familial HDL deficiency syndrome, or other cardiovascular diseases derived from low levels of HDL will not benefit from therapy aimed at lowering plasma LDL levels.

  The problems with current therapies for cardiovascular disease stem in part from the fact that the biology involved in intracellular and extracellular cholesterol migration is not well understood. Furthermore, the proteins that play a role in cholesterol migration are not well known. Accordingly, there remains a need for methods of treating humans suffering from cholesterol cell biology and other disorders associated with cardiovascular disease and hypercholesterolemia. In addition, there remains a need for new methods of diagnosing cardiovascular disease and new methods of screening patients to identify those at high risk for developing cardiovascular disease.

  The identification of genes and proteins involved in cholesterol transport will be useful in the development of pharmaceutical agents for the treatment of heart disease and other disorders associated with hypercholesterolemia and atherosclerosis. In addition, the identification of such genes would be useful in the development of screening assays that screen for compounds that modulate the expression of genes associated with cholesterol transport. Identification of such modulatory compounds will be useful in the development of additional therapeutic agents. Furthermore, the identification of genes and proteins involved in cholesterol transport may be useful as a diagnostic indicator of other disorders associated with cardiovascular disease and hypercholesterolemia.

  The present invention provides novel polypeptides and polynucleotides involved in cholesterol efflux. Specifically, the present invention provides novel ATP-binding cassette (ABC1) polypeptides and novel polynucleotides that encode ABC1 polypeptides. The terms “ABC1” and “ABCA1” are alternative names for the same ATP-binding cassette protein and gene. The present invention provides ABC1 polypeptides, polypeptide fragments and polypeptide variants. In one preferred embodiment, the present invention provides an isolated polypeptide comprising SEQ ID NO: 2. In another preferred embodiment, the present invention provides an isolated polypeptide comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 2. The present invention also provides ABC1 polypeptides from Tangier disease patients. In one preferred embodiment, the present invention provides an isolated polypeptide comprising SEQ ID NO: 8. In another preferred embodiment, the present invention provides an isolated polypeptide comprising SEQ ID NO: 10.

  In addition, the present invention provides ABC1 polynucleotides, polynucleotide fragments and polynucleotide variants. In one preferred embodiment, the present invention provides an isolated polynucleotide encoding a polynucleotide comprising SEQ ID NO: 2. In another preferred embodiment, the present invention provides an isolated polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 2. In another preferred embodiment, the invention also relates to an isolated polynucleotide comprising a nucleotide sequence complementary to a polynucleotide encoding a polypeptide comprising SEQ ID NO: 2 or at least 98 to SEQ ID NO: 2. An isolated polynucleotide comprising a nucleotide sequence complementary to a polynucleotide encoding a polypeptide comprising an amino acid sequence having% identity is provided.

  In another preferred embodiment, the present invention provides an isolated ABC1 polynucleotide comprising SEQ ID NO: 1. In a further preferred embodiment, the present invention provides an isolated polynucleotide comprising nucleotides 291-7074 of SEQ ID NO: 1. More preferably, the polynucleotide comprises a nucleotide sequence having at least 95% identity with SEQ ID NO: 1. In other more preferred embodiments, the polynucleotide comprises a nucleotide sequence having at least 96%, 97%, 98% or 99% identity to SEQ ID NO: 1. In another preferred embodiment, the present invention also relates to an isolated polynucleotide comprising a nucleotide sequence complementary to a polynucleotide comprising SEQ ID NO: 1, a polynucleotide comprising nucleotides 291-7074 of SEQ ID NO: 1 and An isolated polynucleotide comprising a complementary nucleotide sequence and an isolated polynucleotide complementary to a polynucleotide comprising a polynucleotide sequence having at least 90% identity to SEQ ID NO: 1 are provided.

  The present invention also provides ABC1 polynucleotides corresponding to the 5 'flanking region of the ABC1 gene. In one preferred embodiment, the present invention provides an isolated polynucleotide comprising SEQ ID NO: 3. In other preferred embodiments, the present invention provides an isolated polynucleotide comprising nucleotides 1-1532, 1080-1643, 1181-11643, 1292-1643, or 1394-1532. Preferably, the isolated polynucleotide comprises nucleotides 1394-1532 of SEQ ID NO: 3. In another preferred embodiment, the present invention provides an isolated polynucleotide that hybridizes under stringent conditions to a polynucleotide comprising SEQ ID NO: 3. In another preferred embodiment, the invention also provides an isolated hybridized under stringent conditions to a polynucleotide comprising nucleotides 1-1532, 1080-1043, 1181-11643, 1292-1643, or 1394-1532. A polynucleotide is provided. In yet another preferred embodiment of the invention, an isolated polynucleotide is provided that has at least 80% identity to the polynucleotide comprising SEQ ID NO: 3. More preferably, the polynucleotide has at least 90% identity to the polynucleotide comprising SEQ ID NO: 3. Even more preferably, the polynucleotide has at least 95% identity to the polynucleotide comprising SEQ ID NO: 3. Also provided in preferred embodiments are isolated polynucleotides having at least 80% identity to a polynucleotide comprising nucleotides 1-1532, 1080-1643, 1181-1634, 1292-1643, or 1394-1532. Is done. More preferably, the polynucleotide is at least 90% identical to a polynucleotide comprising nucleotides 1-1532, 1080-1643, 1181-1634, 1292-1643, or 1934-1532 of SEQ ID NO: 3, even more preferably Has 95% identity. In addition, the present invention provides an isolated polynucleotide comprising a nucleotide sequence complementary to the 5 'flanking ABC1 polynucleotide described above. In one preferred embodiment, the present invention provides an isolated polynucleotide comprising a nucleotide sequence that is complementary to a polynucleotide comprising SEQ ID NO: 3. In another preferred embodiment, the invention comprises a single nucleotide sequence that is complementary to a polynucleotide comprising nucleotides 1-1532, 1080-1643, 1181-1634, 1292-1643, or 1394-1532 of SEQ ID NO: 3. A released polynucleotide is provided.

  The present invention also provides ABC1 polynucleotides corresponding to the 3 'flanking region of the ABC1 gene. In a preferred embodiment, the present invention provides an isolated polynucleotide comprising SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, and its complementary sequence. In another preferred embodiment, the invention provides an isolated polynucleotide that hybridizes under stringent conditions to a polynucleotide comprising SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and its complementary sequence. provide. In yet another preferred embodiment, the invention provides an isolated polynucleotide having at least 80% identity to a polynucleotide comprising SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6. More preferably, the polynucleotide has at least 90% identity to a polynucleotide comprising SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6. Even more preferably, the polynucleotide has at least 95% identity to the polynucleotide comprising SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.

  In addition, the present invention provides ABC1 polynucleotides from patients with Tangier disease. In one preferred embodiment, the present invention provides an isolated polynucleotide encoding a polypeptide comprising SEQ ID NO: 8. In another preferred embodiment, the present invention comprises a single polynucleotide comprising SEQ ID NO: 7. A released polynucleotide is provided. In yet another embodiment, the present invention provides an isolated polynucleotide encoding a polypeptide comprising SEQ ID NO: 10. In yet another preferred embodiment, the present invention provides an isolated polynucleotide comprising SEQ ID NO: 9. The present invention further provides an isolated polynucleotide comprising a nucleotide sequence having complementarity to the described polynucleotide.

  In another aspect, the present invention provides a composition comprising any of the polynucleotides described above and a suitable carrier. In one preferred embodiment, the invention provides an isolated polynucleotide encoding a polypeptide comprising SEQ ID NO: 2, a polynucleotide comprising SEQ ID NO: 1, a polynucleotide comprising nucleotides 291-7074 of SEQ ID NO: 1. Provided is a composition comprising a nucleotide, a polynucleotide encoding a polynucleotide comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 2, and a suitable carrier. In another preferred embodiment, the composition comprises an isolated polynucleotide comprising a nucleotide sequence having at least 90% identity with a polynucleotide comprising SEQ ID NO: 1 and a suitable carrier. In other preferred embodiments, the composition comprises an isolated polynucleotide comprising SEQ ID NO: 3 or nucleotides 1-1532, 1080-1643, 1181-1634, 1292-1643 or 1394-1532 of SEQ ID NO: 3. Including isolated polynucleotides and suitable carriers. In still other preferred embodiments, the present invention relates to polynucleotides that hybridize under stringent conditions to the polynucleotide of SEQ ID NO: 3, or nucleotides 1-1532, 1080-1643, 1181-11643, 1292 of SEQ ID NO: 3. -1643, a composition comprising a polynucleotide comprising 1394-1532 and a polynucleotide having at least 80% identity to a polynucleotide comprising SEQ ID NO: 3, or nucleotides 1-1532, 1080- of SEQ ID NO: 3 Compositions comprising a polynucleotide comprising 1643, 1181-11643, 1292-1643 or 1394-1532 and a suitable carrier are provided. Also provided by the present invention is a composition comprising an isolated polynucleotide comprising a nucleotide sequence complementary to any of the aforementioned polynucleotides and a suitable carrier.

  In addition, the present invention provides recombinant vectors and host cells comprising any of the ABC1 polynucleotide sequences described above. In one preferred embodiment, the present invention provides an isolated polynucleotide encoding a polynucleotide comprising SEQ ID NO: 2, an isolated polynucleotide comprising SEQ ID NO: 1, nucleotide 291- of SEQ ID NO: 1 Provided is a recombinant vector comprising an isolated polynucleotide comprising 7074 or an isolated polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 2. In another preferred embodiment, the recombinant vector comprises an isolated polynucleotide comprising a nucleotide sequence having at least 90% identity, more preferably 95% identity to a polynucleotide comprising SEQ ID NO: 1. In yet another preferred embodiment, the recombinant vector comprises an isolated polynucleotide comprising SEQ ID NO: 7 or SEQ ID NO: 9. The present invention further provides a recombinant vector comprising an isolated polynucleotide comprising a nucleotide complementary to any of the aforementioned polynucleotides. In yet another preferred embodiment, the recombinant vector comprises any of the polynucleotides described above and further comprises a heterologous promoter polynucleotide. One suitable heterologous promoter is the cytomegalovirus promoter. In a particularly preferred embodiment, the recombinant vector is pCEPhABC1.

  The present invention also provides a recombinant vector comprising an isolated polynucleotide comprising the ABC1 5 'flanking sequence. In one preferred embodiment, the invention provides an isolated polynucleotide comprising SEQ ID NO: 3 or nucleotides 1-1532, 1080-1643, 1181-1634, 1292-1643, or 1394-1532 of SEQ ID NO: 3. A recombinant vector comprising an isolated polynucleotide comprising is provided. Furthermore, in other preferred embodiments, the present invention relates to polynucleotides that hybridize under stringent conditions to the polynucleotide of SEQ ID NO: 3, or nucleotides 1-1532, 1080-1643, 1181-1634 of SEQ ID NO: 3. Recombinant vectors comprising polynucleotides comprising 1292-1643, or 1394-1532, as well as recombinant vectors comprising polynucleotides having at least 80% identity to these polynucleotides are provided. The present invention further provides a recombinant vector comprising an isolated polynucleotide comprising a nucleotide sequence complementary to any of the polynucleotides described above. In yet another preferred embodiment, the recombinant vector comprises any of the described polynucleotides and further comprises at least one polynucleotide encoding a heterologous polypeptide. Suitable heterologous polypeptides include luciferase, β-galactosidase, chloramphenicol acetyltransferase transferase, and green fluorescent protein. Preferably the heterologous polypeptide is a luciferase protein. In a particularly preferred embodiment, the recombinant vector is pAPR1.

  In addition, the present invention provides host cells comprising any of the recombinant vectors described above. The present invention further provides a composition comprising the above-described recombinant vector and a suitable carrier.

  The present invention also provides a method for producing ABC1 protein in mammalian host cells and a method for expressing ABC1 protein in mammalian subjects. A method of producing ABC1 protein in a mammalian host cell comprises (a) using a recombinant expression vector comprising a sufficient amount of a polynucleotide encoding ABC1 to produce a detectable level of ABC1 protein. Transfection and then (b) purifying the produced ABC1 protein. A method for expressing ABC1 protein in a mammalian subject comprises administering to the mammalian subject a recombinant expression vector comprising a sufficient amount of a polynucleotide encoding ABC1 to express ABC1 protein in the mammalian subject.

In addition, the present invention provides compositions and methods suitable for increasing cholesterol efflux from cells of a mammalian subject. In one preferred embodiment, the method comprises administering to the mammalian subject a recombinant expression vector comprising a sufficient amount of a polynucleotide encoding ABC1 to increase cholesterol efflux from the cell. Suitably the recombinant expression vector comprises an isolated polynucleotide encoding a polypeptide comprising SEQ ID NO: 2, an isolated polynucleotide comprising SEQ ID NO: 1, nucleotides 291-7074 of SEQ ID NO: 1 A vector comprising an isolated polynucleotide and an isolated polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 2. Preferred expression vectors include viral vectors, particularly adenoviral vectors and lentiviral vectors. In another embodiment, the present invention is, DNA-ligand complexes, adenovirus - ligand -DNA complexes, direct injection of DNA, non including CaPO ₄ precipitation, gene gun techniques, electroporation, liposomes and lipofection - A viral delivery system is provided.

  In another preferred embodiment, the method of increasing cholesterol efflux from cells of a mammalian subject comprises administering to the mammalian subject a therapeutic amount of a compound that increases ABC1 expression in the cells. One suitable method involves administering a cAMP analog to a mammalian subject. Suitable cAMP analogs include 8-bromo cAMP, N6-benzoyl cAMP, and 8-thiomethyl cAMP. Another suitable method involves administering to a mammalian subject a compound that increases cAMP synthesis, such as forskolin. Yet another suitable method involves administering to a mammalian subject a compound that inhibits the degradation of cAMP, such as a phosphodiesterase inhibitor. Suitable phosphodiesterase inhibitors include rolipram, theophylline, 3-isobutyl-1-methylxanthine, R020-1724, vinpocetine, zaprinast, dipyridamole, milrinone, amrinone, pimobendan, cilostamide, enoximone, peroximone and vesnarinone.

  In addition, another suitable method for increasing cholesterol efflux from cells of a mammalian subject is to administer to the mammalian subject at least one ligand for a nuclear receptor in an amount sufficient to increase cholesterol efflux. including. Suitable ligands include LXR, RXR, FXR, SXR and PPAR ligands. In one preferred embodiment, the method comprises administering to a mammalian subject a ligand for the LXR nuclear receptor. A suitable LXR ligand is 20 (S) hydroxycholesterol. 22 (R) hydroxycholesterol, 24 (S) hydroxycholesterol, 25-hydroxycholesterol, and 24 (S), 25 epoxycholesterol. Preferably, the LXR ligand is 20 (S) hydroxycholesterol. In another preferred embodiment, the method comprises administering to a mammalian subject a ligand for the RXR nuclear receptor. Suitable RXR ligands include 9-cis retinoic acid, retinol, retinal, all-trans retinoic acid, 13-cis retinoic acid, acitretin, fenretinide, etretinate, CD495, CD564, TTNN, TTNPNP, TTAB, and LGD1069. Preferably, the RXR ligand is 9-cis retinoic acid. In another preferred embodiment, the method comprises administering to a mammalian subject a ligand for the PPAR nuclear receptor. One suitable ligand is a ligand selected from the class of thiazolidinediones. In yet another preferred embodiment, the method comprises administering at least two ligands for nuclear receptors. In particularly preferred embodiments, the ligand is 20 (S) hydroxycholesterol and 9-cis retinoic acid.

  In addition, another suitable method for increasing cholesterol efflux from cells of a mammalian subject includes administering to the mammalian subject an amount of eicosanoid sufficient to increase cholesterol efflux. Suitable eicosanoids include prostaglandin E2, prostaglandin J2, and prostacyclin (prostaglandin I2).

  In another embodiment, the invention provides a method for administering to a mammalian subject an amount of a compound that increases ABC1 activity sufficient to increase cholesterol efflux from the cell of the mammalian subject. Provides a way to increase cholesterol efflux.

  The present invention also provides a method suitable for increasing ABC1 gene expression in a mammalian subject. In one preferred embodiment, the method comprises administering to the mammalian control an amount of at least one ligand for the nuclear receptor sufficient to increase ABC1 gene expression. Suitable ligands include ligands for LXR, RXR, FXR, SXR and DPAR nuclear receptors. In another preferred embodiment, the method comprises administering to the mammalian subject an amount of a cAMP analog sufficient to increase ABC1 gene expression. In yet another preferred embodiment, the method comprises administering to a mammalian subject an amount of a compound that increases the synthesis of cAMP sufficient to increase ABC1 gene expression.

  In addition, the present invention provides that (a) the reporter cDNA is operably linked with an expression modulating portion of a mammalian abc1 gene to generate a recombinant reporter construct; (b) the recombinant reporter construct is hosted Transfecting a population of cells; (c) assaying the level of reporter gene expression in a sample of host cells; (d) contacting the test compound to be screened with the host cell; (e) contacting the test compound. Followed by assaying the level of reporter gene expression in a sample of the host cell; then (f) comparing the relative change in the level of reporter gene expression caused by exposure to the test compound, whereby ABC1 expression A method for screening a test compound for ABC1 expression modulation activity, comprising the step of measuring the modulation activity To provide. The recombinant reporter construct comprises a reporter gene operably linked to an expression modulating portion of a mammalian ABC1 gene, such as any of the ABC1 5 'flanking region sequences provided by the present invention. In one preferred embodiment, the expression modulating portion of the ABC1 gene comprises SEQ ID NO: 3. In another preferred embodiment, the expression modulating portion of the ABC1 gene comprises nucleotides 1-1532, 1080-1643, 1181-1634, 1292-1643, 1394-1643, or 1394-1532 of SEQ ID NO: 3. Suitable reporter cDNAs include luciferase, β-galactosidase, chloramphenicol acetyltransferase, and green fluorescent protein cDNA. Preferably, the host cell is a mammalian cell. In a particularly preferred embodiment of the method, the recombinant reporter construct is pAPR1.

  Also provided in the present invention is (a) assaying the level of cholesterol efflux in a sample of mammalian cells maintained during culture to determine a control level of cholesterol efflux; (b) Contacting with the test compound to be screened; (c) assaying the level of cholesterol efflux in the sample of cells after contact with the test compound; (d) ABC1-mediated cholesterol efflux in the sample of cells after contact with the test compound; Comprising: determining whether the test compound promotes ABC1-culture cholesterol efflux from cells in culture, whereby the test compound is isolated from cells in culture. ABC1-Test compound is screened to determine whether it promotes cultured cholesterol efflux. It is a method of training. The cells can be derived from primary cultures or cell lines. Suitable cells for screening test compounds include fibroblasts, macrophages, liver, and intestinal cell lines. Preferably, the cell line is RAW 264.7. In one preferred embodiment, ABC1-mediated cholesterol efflux is measured using an anti-ABC1 antibody that inhibits the activity of ABC1 upon binding. In another preferred embodiment, ABC1-mediated cholesterol efflux is measured using an antisense ABC1 polynucleotide. In a particularly preferred embodiment, the antisense polynucleotide comprises SEQ ID NO: 57.

  In addition, the present invention provides a method for detecting a comparative level of ABC1 expression in cells of a mammalian subject. Such methods can be used to measure a subject's susceptibility to coronary heart disease. A method is provided for detecting a comparative level of ABC1 expression in a mammalian subject cell, comprising: (a) obtaining a cell sample from the mammalian subject; and (b) assaying the level of ABC1 mRNA expression in the cell sample; Then, (c) comparing the level of ABC1 mRNA expression in the cell sample with a predetermined standard level of ABC1 mRNA expression, thereby detecting a comparative level of ABC1 gene expression in the cells of the mammalian subject. Suitable methods for measuring the level of ABC1 mRNA expression include, for example, RT-PCR, Northern blots, and RNAse protection assays.

  The present invention also provides a method for detecting a comparative level of ABC1 protein in cells of a mammalian subject. Such methods can be used to measure a subject's susceptibility to coronary heart disease. A method is provided for detecting a comparative amount of ABC1 protein in a mammalian subject cell, comprising: (a) obtaining a cell sample from the mammalian subject; (b) assaying the amount of ABC1 protein in the cell sample; (C) comparing the amount of ABC1 protein in the cell sample with a predetermined standard amount of ABC1 protein, thereby detecting a comparative level of ABC1 protein in the cells of the mammalian subject. The amount of ABC1 protein can be measured using various immunoassays available in the art. For example, the amount of ABC1 protein can be measured by (a) contacting a cell sample with a population of anti-ABC1 antibodies and then detecting (b) a specific-binding ABC1 antibody associated with the sample. . Suitable methods for detecting ABC1 antibody include western blotting, immunoprecipitation and FACS.

  In another aspect, the present invention provides an antibody that specifically binds to the described ABC1 polypeptide. In one preferred embodiment, the present invention provides an isolated antibody that specifically binds to an isolated polypeptide comprising SEQ ID NO: 2. In another preferred embodiment, the present invention provides an isolated antibody that specifically binds to an isolated polypeptide comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 2. The antibody can be a monoclonal antibody, or the antibody can be a polyclonal antibody. In yet another embodiment, the antibody inhibits the cholesterol transport activity of ABC1 polypeptide upon binding to ABC1 polypeptide.

  In addition, the present invention comprises a compound ABC1 comprising a reporter cDNA operably linked to an expression modulating portion of a mammalian ABC1 gene in a sufficient amount in at least one assay and instructions for use Kits suitable for screening compounds for measuring expression modulation activity are provided. In one preferred embodiment, the kit further comprises means for detecting a reporter gene. In another preferred embodiment, the expression modulating portion of the mammalian ABC1 gene comprises SEQ ID NO: 3. In yet another preferred embodiment, the expression modulating portion of the mammalian ABC1 gene comprises nucleotides 1-1532, 1080-1643, 1181-1634, 1292-1643, 1394-1643, or 1394-1532 of SEQ ID NO: 3. . Suitable reporter cDNAs include luciferase, β-galactosidase, chloramphenicol acetyltransferase and green fluorescent protein cDNA. Preferably the reporter cDNA is luciferase. In a particularly preferred embodiment of the method, the recombinant reporter construct is pAPR1.

  The present invention also provides kits suitable for screening compounds to determine whether the compounds modulate ABC1-dependent cholesterol efflux. In one preferred embodiment, the kit comprises an amount of inactivated anti-ABC1 antibody sufficient for at least one assay and instructions for use. In another preferred embodiment, the kit comprises an amount of antisense ABC1 oligonucleotide sufficient for at least one assay and instructions for use. In a particularly preferred embodiment, the antisense ABC1 oligonucleotide comprises SEQ ID NO: 53.

  According to the present invention, novel polypeptides that increase cholesterol efflux from cells are provided. In particular, the present invention provides novel ATP-binding cassette 1 (ABC1) polypeptides that have been shown to increase cholesterol efflux.

  ABC1 is a member of a family of ATP-binding cassette proteins that are present in the cell membrane and transport a wide variety of substrates across the plasma membrane using ATP hydrolysis. It should be noted that the terms “ABC1” and (ABCA1) both refer to the same ATP-binding cassette protein. The term “ABCA1” was introduced in 1999 by the naming committee and has received limited acceptance in the art. To date, more than 30 members of this family have been identified in the human genome. These homologous proteins contain a channel-like structure through which molecules are transported across the cell membrane and one or more domains that are coupled to ATP to couple energy-generated ATP-hydrolysis to transport. Members of the family are multidrug resistance factors (MDR / P glycoprotein; Chen et al., Cell, 47: 381-389 (1986); Stride et al., Mol. Pharmacol., 49: 962-971 (1996)), antigens Transporters associated with presentation (Nefjes et al., Science, 261: 769-771 (1993); Shepherd et al., Cell, 74: 577-584 (1993)), and cystic fibrosis transmembrane conductance regulator (Chang et al., J Biol.Chem., 269: 18572-18575 (1994); Rommens et al., Science, 245: 1059-1065 (1989)). ABC transporter family members generally consist of four domains found in two symmetrical halves connected by a long charged region and a highly hydrophobic segment. Each half contains a hydrophobic domain containing 6 transmembrane segments and a hydrophilic nucleotide binding domain containing highly conserved Walker A and B sequence motifs typical of many ATPases (Hyde et al., Nature, 346: 362-365 (1990); Luciani et al., Genomics, 21: 150-159 (1994)). The activity of the transporter depends on its interaction with ATP in the nucleotide binding domain and by regulation via phosphorylation of the residue in the region connecting the two symmetric halves (Becq et al., J. Biol. Chem., 272: 2695). -2699 (1997)).

  Several lines of evidence described herein have identified ABC1 as a very important protein in apolipoprotein-mediated mobilization of intracellular cholesterol stores. First, the studies presented here have shown that ABC1 is deficient in Tangier disease, a genetic disorder characterized by HDL-cholesterol metabolism. As already shown and shown in Example 1 herein, genetic defects in Tangier disease cause defects in the pathway of apolipoprotein-mediated efflux of cholesterol from within the cell, resulting in significantly reduced cholesterol efflux activity and It results in low HDL-cholesterol levels (Oram et al., J. Lipid Res., 37: 2743-2491 (1996); Francis et al., J. Clin. Invest., 96: 78-87 (1995)). Genetic linkage analysis of families with Tangier disease assigned defective genes to intervals on chromosome 9q31 (Rest et al., Nature Genetics, 20: 96-98 (1998)). Public database searches reveal that the ABC1 gene is located on chromosome 9q22-9q31, which includes, but is wider than, the interval revealed by Rust et al. (Luciani et al., Supra (1994)). Based on that data, irradiation hybrid mapping of the human ABC1 gene was performed, which located the gene between two markers clearly within the 7-cM region of human chromosome 9q31 reported by Rust et al. In addition, as shown in Example 2, microarray analysis revealed that the ABC1 gene was under-expressed 2.5-fold in Tangier patient cells compared to normal cells. These studies identified the defective gene in Tangier disease as ABC1. In addition, the further work presented here linked ABC1 activity to cholesterol efflux activity. First, studies have shown that inhibitors of ABC1 transport activity such as 4,4-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and sulfobromophthalein (BSP) are also apo AI from fibroblasts. -Demonstrated inhibition of mediated cholesterol efflux (see Example 6). Also, using antisense ABC1 oligonucleotides, inhibition of ABC1 gene expression was shown to inhibit apoAI-mediated cholesterol efflux from fibroblasts (Example 7). In contrast, transfection experiments in which the ABC1 gene was transfected into mouse monocytic cells showed that ABC1 overexpression resulted in increased apoAI-mediated efflux (Example 8). Finally, RI-PCR performed with wild-type and Tangier patient mRNA is prepared by a cellular state associated with cholesterol efflux in normal skin fibroblasts where ABC1 mRNA expression is normal, but in Tangier patient fibroblasts It was clarified that this was not the case (Example 9). Based on these findings, ABC1 was determined to play a major role in cholesterol efflux.

  ABC1 plays a role in the movement of intracellular cholesterol to the outer leaflet of the plasma membrane. Absence of ABC1 or deficient transport of intracellular cholesterol by ABC1 results in a lack of cholesterol in specific membrane domains with which it specifically interacts with apoAI and other apolipoproteins (Stangl et al., J. Biol Chem., 273: 31002-31008 (1998); Babitt et al., J. Biol. Chem., 272: 13242-13249 (1997)). Failure to deliver cholesterol to apoAI leads to the formation of cholesterol-deficient HDL particles that are rapidly cleared from the blood (Bojanovski et al., J. Clin. Invest., 80: 1742-1747 (1987)).

(Definition)
The following definitions are provided to facilitate understanding of certain terms used throughout this specification.

  For the purposes of the present invention, “isolated” refers to a material that has been removed from its original environment (eg, the natural environment if it is naturally occurring), thus “ “Modified by the hand of man”. For example, an isolated polynucleotide can be part of a vector or composition, or can be contained within a cell and still “isolated”. This is because the vector, composition or specific cell is not the original environment of the polynucleotide.

  As used herein, “polynucleotide” is defined to include DNA and RNA of both synthetic and natural origin. The polynucleotide can exist as single-stranded or double-stranded DNA or RNA, or RNA / DNA heteroduplex. Thus, a polynucleotide of the invention can consist of any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA. For example, a polynucleotide can be single stranded and double stranded DNA, single stranded and double stranded regions, or a mixture of single stranded, double stranded and triple stranded regions, single stranded, double stranded. RNA, which can be RNA and RNA that is a mixture of single and double stranded regions, single stranded, or more typically double or triple stranded, or a mixture of single and double stranded regions, and It can consist of a hybrid molecule containing RNA. In addition, a polynucleotide can consist of triple-stranded regions comprising RNA or DNA or both RNA and DNA. Polynucleotides can also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. Various modifications can be made to DNA and RNA; thus, “polynucleotide” includes chemically enzymatically or metabolically modified forms of a polynucleotide. The term “polynucleotide” also includes short polynucleotides, often referred to as oligonucleotides.

  The term “polypeptide” refers to any peptide or protein comprising two or more amino acids linked together by peptide bonds or modified peptide bonds. “Polypeptide” refers to both short amino acid sequences commonly referred to as peptides, as well as longer amino acid sequences commonly referred to as proteins. Polypeptides can contain amino acids other than those encoded by the 20 genes. In addition, the polypeptides can be modified by natural processes such as processing and other post-translational modifications, or by chemical modification techniques well known in the art. A given polypeptide can contain many types of modifications. Also, the same type of modification may be present in the same or varying degrees at one or more sites in the polypeptide. Modifications can occur anywhere in the polypeptide including the peptide backbone, amino acid side chains and the amino or carboxyl terminus. Modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, formylation, gamma-carboxylation, glycosylation, hydroxylation, iodination, methylation, myristoylation, oxidation, Includes phosphorylation, prenylation, sulfation, selenoylation, and covalent attachment of nucleotides or nucleotide derivatives, lipids or lipid derivatives, or phosphatidylinositol. Other modifications include t-RNA-mediated loading of amino acids such as cross-linking, cyclization, pyroglutamate formation, GPI anchor formation, proteolytic processing racemization, and arginylation and ubiquitination. For example, Proteins-Structure and Molecular Properties, 2nd edition, T.A. E. Creighton, W.M. H. Freedman and Co. , New York (1993); , Posttranslational Protein Modification: Perspectives and Prospects, in Posttranslational Covalent Modification of Proteins. B, C.I. Johnson. Ed. , Academic Press, New York (1983); Seifter et al. , Meth. Enzymol. 182: 626-646 (1990); and Rattan et al. , Protein Synthesis: Poststranlational Modification and Aging, Ann. N. Y. Acad. Sci. 663: 48-62 (1992)). The polypeptides of the present invention can be prepared by any suitable method. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for producing such polypeptides are well known in the art.

  The “polynucleotide” of the present invention is SEQ ID NO: 3 or nucleotides 1-1532, 1080-1643, 1181-1634, 1292-1643, 1394-1643 or 1394-1532 of SEQ ID NO: 3, or their complements. Polynucleotides that can hybridize under stringent hybridization conditions. The polynucleotides of the present invention also include polynucleotides that can hybridize under stringent hybridization conditions to SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 or their complements.

  “Strict hybridization conditions” include 50% formamide, 5 × SSC (750 mM NaCl, 75 mM sodium citrate), mM sodium phosphate (pH 7.6), 5 × Denhardt's solution, 10% dextran sulfate, and Refers to overnight incubation at 42 ° C. in a solution containing 20 μg / ml modified sheared salmon sperm DNA, followed by washing the filter in 0.1 × SSC at about 65 ° C.

  As used herein, the term “complementarity” refers to, for example, primer binding between two strands of a double-stranded polynucleotide or on an oligonucleotide primer and a single-stranded polynucleotide to be amplified or sequenced. Refers to hybridization or base pairing between nucleotides, such as between sites. Two single-stranded nucleotide molecules are complementary if the nucleotides of one strand are optimally aligned with appropriate nucleotide insertions, deletions or substitutions, pairs at least about 80% of the nucleotides of the other strand It is said.

  “Identity” as known in the art is a relationship between two or more polynucleotide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. Also, “identity” or “similarity” has been recognized in the art to refer to the degree of sequence relatedness between polypeptide or polynucleotide sequences, as determined by matches between strings of such sequences. Has meaning. "Identity" and "Similarity" are (Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, (1988); Biocomputing: Informatics and Genome Projects. , Ed., Academic Press, New York, (1993); Computer Analysis of Sequence data, Part I, Griggin, AM, and Griffin, H. G., Edit, Humana 94, Newa; Sequence Analysis in Molecular Biology, vo Heinje, G., Academic Press, (1987); and Sequence Analysis Primer, Gribskov, M. and Devereux, J., Editing, M Stockton Press, New York, Ar, D, H, and 1991; .. can be calculated using a number of well-known methods, including those published in SIAM J Applied Math 48: 1073 (1988). Methods commonly used to determine identity or similarity between two sequences are not limited, but are described in “Guide to Huge Computers,” Martin J. et al. Bishop, ed. , Academic Press, San Diego, (1994), and Carillo, H .; Lipton, D .; , SIAM J Applied Math 48: 1073'1988). Preferred methods for determining identity are designed to give the greatest match between the sequences tested. Methods for aligning polynucleotides or polypeptides are described in the GCG program package (Devereux, J., R, Nucleic Acids Research (1984) 12 (1): 387 (1984)), BLASTP, BLASTIN, FASTA (Atschul, S F. et al., J. Molec.Biol. 215: 403 (1990). , A edits in Applied Mathematics 2: 482-489 (1981)).

  When using any of the sequence alignment programs to determine whether a particular sequence is, for example, 90% identical to a reference sequence, the parameter is the percentage of identity of the reference polypeptide or polynucleotide. Calculated over the entire length and set to allow gaps in identity up to 10% of the total number of nucleotides in the reference polynucleotide.

  A preferred method for determining the best overall match between a query sequence (sequence of the invention) and subject sequence, also referred to as global sequence alignment, is described by Brutlag et al. (Comp. App. Biosci. 6: 237-245 (1990)). Can be determined using a FASTDB computer program based on this algorithm. The term “sequence” includes nucleotide and amino acid sequences. In sequence alignment, the query and subject sequences are both nucleotide sequences or both amino acid sequences. The result of the global sequence alignment is expressed as percent identity. Preferred parameters used in the FASTDB search of DNA sequences for calculating percent identity are: matrix = unitary, k-tuple = 4, mismatch penalty = 1, junction penalty = 30, randomization base length = 0, and cut-off score = 1. Gap penalty = 5, gap size penalty 0.05, and window size = 500 or how short is the length of the query sequence in nucleotide bases. Preferred parameters used to calculate percent identity and similarity of amino acid alignments are: matrix = PAM 150, k-tuple = 2, mismatch penalty = 1, junction penalty = 20, randomization group length = 0, Cut-off score = 1, gap penalty = 5, gap size penalty = 0.05, and window size = 500 or how short is the length of the query sequence in amino acid residues.

  By way of illustration, a polynucleotide having a nucleotide sequence that is at least 90% “identical” to the sequence contained in SEQ ID NO: 1 is a polynucleotide sequence up to 10 for each 100 nucleotides of the total length of SEQ ID NO: 1. Means that the nucleotide sequence of the polynucleotide is identical to the sequence contained in SEQ ID NO: 1. In other words, to obtain a polynucleotide comprising a nucleotide sequence having at least 90% identity to SEQ ID NO: 1, up to 10% of the nucleotides in the sequence contained in SEQ ID NO: 1 are deleted and inserted Or can be replaced with other nucleotides. These changes can occur anywhere in the entire polynucleotide and can be individually dispersed within one or more consecutive groups within the nucleotide or within SEQ ID NO: 1.

  Similarly, a polypeptide having an amino acid sequence that is at least 98% “identical” to the sequence contained in SEQ ID NO: 2 may have a polypeptide sequence up to 2 for each 100 amino acids in the total length of SEQ ID NO: 2. It means that the amino acid sequence of the polypeptide is identical to the sequence contained in SEQ ID NO: 2, except that it can contain amino acid modifications. In other words, in order to obtain a polypeptide having an amino acid sequence that is at least 98% identical to SEQ ID NO: 2, up to 2% of the amino acid residues in the sequence contained in SEQ ID NO: 2 are deleted and inserted. Or can be replaced with other amino acid residues. These changes can occur anywhere in the entire polypeptide and can be dispersed individually between residues or in one or more consecutive groups within SEQ ID NO: 2.

  A “polypeptide having biological activity” is an activity that is similar but not necessarily identical to the activity of a polypeptide of the invention, as measured in a particular biological assay, with or without dose dependence. Where there is a dose dependency that refers to a polypeptide that exhibits (eg, cholesterol transport activity), it need not be identical to that of the polypeptide, but rather is given in comparison to the polypeptide of the invention. Is substantially similar to the dose-dependence in the activity produced (eg, the candidate polypeptide has a greater activity or less than about 25-fold, preferably less than about 10-fold less activity than the polypeptide of the invention) Most preferably will exhibit an activity about 3 times less).

  “Polypeptide variant” refers to a polypeptide that differs from the ABC1 polypeptide of the invention but retains its essential properties. In general, the variants are generally closely similar and are identical to the polypeptide comprising SEQ ID NO: 2 in many regions. Preferably, the polypeptide variant retains biological activity, ie cholesterol transport activity. Variants include, but are not limited to, splice variants and allelic variants and addition, deletion and substitution variants.

  Similarly, a “polynucleotide variant” refers to a polynucleotide that differs from the polynucleotide of the invention but retains its essential properties. The variant can include modifications in the coding region, non-coding region, or both. Thus, for example, the ABC1 polynucleotide variant has a nucleotide sequence that encodes a polypeptide having cholesterol transport activity, but different from that of SEQ ID NO: 1. Also, for example, a polynucleotide variant has a nucleotide sequence that differs from that of SEQ ID NO: 3, but retains promoter activity. Particularly preferred are polynucleotide variants that contain modifications that produce silent substitutions, additions or deletions, but do not alter the properties or activities of the encoded polypeptide. Nucleotide variants generated by silent substitution are preferred due to the degeneracy of the genetic code. Furthermore, variants in which 10-20, 5-10, 1-5 or 1-2 amino acids are substituted in any combination and deleted or added are also preferred. Polynucleotide variants can be generated for a variety of reasons, eg, to optimize codon expression for a particular host (eg, those codons in human mRNA that are preferred by bacterial hosts such as E. coli). Change to).

  An “allelic variant” is a naturally occurring variant that refers to one of several alternative forms of a gene that occupies a given locus on the chromosome of an organism (Genes II, Lewin, B. Ed., John Wiley & Sons, New York (1985)). These allelic variants can vary at either the polynucleotide and / or polypeptide level. Alternatively, non-naturally occurring variants can be generated by mutagenesis techniques or by direct synthesis.

  The term “conservative amino acid substitution” refers to a substitution of a natural amino acid residue with a non-natural residue such that there is little or no amino acid residue polarity or charge at that site. For example, a conservative substitution results from the replacement of a nonpolar residue in a polypeptide with any other non-polar residue. Another example of a conservative substitution is the substitution of an acidic residue with another acidic residue. Conservative substitutions are expected to result in ABC1 polypeptides having functional and chemical characteristics similar to those of naturally occurring ABC1 polypeptides.

  The term “homologous molecular species” refers to polypeptides that correspond to polypeptides identified from different principals. For example, mouse and human ABC1 polypeptides are considered homologous species.

  The term “vector” is used to refer to any molecule (eg, nucleic acid, plasmid, or virus) used to transfer coding information into a host cell.

  The term “expression vector” refers to a vector that is suitable for transformation of a host cell and contains a nucleic acid sequence that directs and / or controls the sequence of an inserted heterologous nucleic acid sequence. Expression includes, but is not limited to, processes such as transcription, translation and RNA splicing, if introns are present.

  As used herein, the term “transcriptional regulatory region” or “expression modulation portion” refers to any region of a gene including, but not limited to, promoters, enhancers and repressors.

  As used herein, the term “promoter” is located upstream (ie, 5 ′) relative to the start codon of a structural gene that controls transcription of the structural gene (generally within about 100 to 1000 bp). An untranscribed sequence.

  As used herein, the term “enhancer” refers to a cis-acting element of DNA, usually 10-300 bp in length, that acts on a promoter to increase transcription. Enhancers are relatively orientation and position independent. They are found 5 'and 3' to the transfer unit.

  A “host cell” is a cell that has been transformed or transfected, or is capable of transformation or transfection by an exogenous polynucleotide sequence. The term includes the progeny of the parent cell, so long as the selected gene is present, regardless of whether the offspring is identical in morphology or genetic makeup to the original parent.

  The term “operably linked” is used herein to refer to an arrangement of flanking sequences in which the flanking sequences thus described are arranged or assembled to perform their normal functions. Thus, a flanking sequence operably linked to a coding sequence can replicate, transcribe and / or translate the coding sequence. For example, a coding sequence is operably linked to a promoter if the promoter can direct transcription of the coding sequence. A flanking sequence need not be adjacent to the coding sequence as long as it functions correctly. Thus, for example, an intervening yet untranslated but transcribed sequence can exist between a promoter sequence and a coding sequence, and the promoter sequence is still considered “operably linked” to the coding sequence. be able to.

  The term “transfection” is used to refer to the uptake of exogenous or exogenous DNA by a cell, and the cell has been transfected if the exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are well known in the art and are disclosed herein. For example, Graham et al., 1973, Virology 52: 456; Sambrook et al., Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratories, 1989); Davis et al., Basic Medicine 6 See Gene 13: 197. Such techniques can be used to introduce one or more exogenous DNA sites into a suitable host cell.

(ABC1 polypeptide)
The present invention relates to novel human ABC1 polypeptides. In one embodiment, the ABC1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2. In contrast to the human ABC1 protein reported by others, the ABC1 polypeptide shown in SEQ ID NO: 2 has an additional 60 amino acids at the amino terminus, which is 2261 amino acids of ABC1 than 2201 amino acids. Proteins are identified (see Langmann et al., In Biochem, Biophys. Res. Comm. 257.20-33 (1999)). In addition, the ABC1 polypeptide shown in SEQ ID NO: 2 differs from other reported sequences in several amino acid residues. Specifically, the current ABC1 polypeptide shown in SEQ ID NO: 2 is K instead of R at residue 159, I instead of V at 765, M instead of I at 823, and T at 1495 I, 1588 has L instead of P, 1914 has K instead of R, and 2108 has L instead of P. To remain consistent with the published record, the amino acid number is that of SEQ ID NO: 2 (Lawn et al., Clin. Insert., 104: R25-31 (1999). As discussed in more detail in, the sequence difference is that the first ABC1 cDNA was cloned from the mouse using a PCR-based strategy and the subsequently reported human ABC1 cDNA sequence was predicted from the sequence of the mouse protein. The ABC1 protein has an approximate molecular weight of 240 kD measured by SDS-PAGE.

  The present invention also relates to an ABC1 polypeptide comprising an amino acid sequence that preferably has at least 98% identity over its entire length to the amino acid sequence of SEQ ID NO: 2. More preferably, the polypeptide has at least 99% identity over its entire length to the amino acid sequence of SEQ ID NO: 2. More preferably, the polypeptide has 100% identity over its entire length to the amino acid of SEQ ID NO: 2. As already defined, the term “identity” refers to the degree of sequence relatedness between polypeptide sequences, which is further defined below.

  Such related ABC1 polypeptides include substitution, deletion, and insertion variants as well as allelic variants, splice variants, fragments, derivatives and homologous molecular species. Preferred polypeptides and polypeptide fragments include polypeptides and fragments that retain the biological activity of ABC1. In particular, polypeptides and fragments that mediate reverse cholesterol transport are preferred. Also preferred are polypeptides and fragments having improved reverse cholesterol transport activity.

  Substitutions, deletions and insertion variants refer to ABC1 polypeptides comprising an amino acid sequence comprising one or more amino acid sequence substitutions, deletions and / or additions compared to the ABC1 amino acid sequence set forth in SEQ ID NO: 2. In preferred embodiments, the variant has 1 to 5, about 1 to 10, or about 1 to 20, or about 1 to 40, or about 1 to 60 amino acid substitutions, additions and / or deletions. For example, the variant can have one or more amino acid residue additions anywhere in the polypeptide and at the carboxyl and / or amino terminus, so long as the variant retains biological function. Also, for example, one or more amino acids can be deleted from any region of the polypeptide including the carboxyl terminus and / or amino terminus without substantial loss of biological function (Ron et al., J. Biol. Biol.Chem., 268: 2984-2988 (1993); Dobeli et al., J. Biotechnology, 7: 199-216 (1988)). As long as the ABC1 variant retains its biological activity, amino acid substitutions can be conservative, non-conservative, or any combination thereof. In addition, substitutions can be made with non-conserved amino acid residues, where the substituted residue may or may not be encoded by the genetic code, and the amino acid residue has been substituted Has a group.

  Suitable variants of ABC1 polypeptide are well known and can be measured using techniques. For example, appropriate ABC1 variants can be determined by identifying regions of the ABC1 molecule that can be altered without destroying biological activity. In addition, as recognized in the art, even regions that may be important for biological activity or structure may be conserved without destroying biological activity or adversely affecting polypeptide structure. Amino acid substitutions can be made. Amino acid residues that can be altered without destroying biological activity can be measured by identifying regions of the ABC1 polypeptide that are not important for activity (Bowie et al., Science, 247: 1306-1310 ( 1990)). For example, ABC1 polypeptides from different species can be compared to determine the amino acid residues and regions of ABC1 molecules that are conserved cross-species. Conserved amino acid residues appear to be important for biological function and / or structure. In contrast, changes in the region of the ABC1 molecule that are not conserved cross-species, and thus are tolerated by natural selection, will not adversely affect biological activity and / or structure. Thus, ABC1 polypeptides with additions, deletions or substitutions in non-conserved regions appear to be suitable variants. Even in relatively conserved regions, chemically similar amino acids can be substituted for naturally occurring residues while retaining activity.

  In addition, structure-function experiments can be used to measure residues in members other than the ATP-binding cassette protein family, such as ABCR and ABC-C, which are important for activity or structure. Such experiments allow the prediction of important amino acid residues in ABC1 variants that correspond to amino acid residues that are important for activity or structure in other ATP-binding cassette proteins. For example, based on structure-function experiments of other ATP-binding cassette proteins, important amino acid residues in ABC1 appear to be found in regions associated with nucleotide binding and sterol transport. Suitable variants include, for example, polypeptides having chemically similar amino acid substitutions in place of such predicted important amino acid residues of ABC1 polypeptide.

  Appropriate ABC1 variants can also be measured using genetic engineering techniques to introduce amino acid changes at specific positions to identify regions of great importance for polypeptide function. Amino acid changes can be made, for example, using site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham et al., Science, 244: 1081-1085 (1989)). The resulting ABC1 variants can then be tested for biological activity using, for example, any one of the cholesterol efflux assays described herein. Variants with specific amino acid residue substitutions that result in disrupted cholesterol efflux activity would not be considered suitable ABC1 variants.

  Additional methods for identifying suitable variants are well known in the art. Furthermore, one skilled in the art will recognize amino acid changes that would be tolerated at certain amino acid positions in the protein (Bowie et al., Supra (1990)). For example, it is generally known that the most buried or internal amino acid residues (within the tertiary structure of a protein) require nonpolar side chains, while fewer features of surface or external side chains are generally conserved. It has been. In addition, allowed conservative amino acid substitutions include death or hydrophobic amino acids Ala, Val, Leu and Ile substitutions, hydroxyl residues Ser and Thr substitutions, acidic residues Asp and Glu substitutions, amide residues Asn and Substitution of Gln, substitution of basic residues Lys, Arg and His, substitution of aromatic residues Phe, Tyr and Trp, substitution of small size amino acids Ala, Ser, Thr, Met and Gly.

  ABC1 variants can be naturally occurring or artificially constructed. Examples of naturally occurring variants are allelic variants and splice variants. Allelic variant refers to one of several alternative forms of a gene occupying a given locus on the chromosome of an organism or population of organisms (Lewin, B., ed., Genes II, John Wiley & Sons). , New York (1985)). Allelic variants can be altered at either the polynucleotide and / or polypeptide level. A splice variant refers to a nucleic acid molecule, usually RNA, that results from RNA transcripts and other processing of intron sequences in the corresponding polypeptide. Alternatively, ABC1 variants can be constructed artificially. For example, ABC1 variants can be constructed using site-directed mutagenesis techniques. Also, for example, an ABC1 variant can be prepared from a corresponding nucleic acid molecule that encodes the variant having a DNA sequence that changes as described from the wild-type DNA sequence set forth in SEQ ID NO: 1.

  The polypeptide fragment refers to a polypeptide comprising less than the full-length amino acid sequence of ABC1 described in SEQ ID NO: 2. Preferred polypeptide fragments include those that retain the biological activity of ABC1. Particularly preferred are fragments that mediate reverse cholesterol transport or have improved reverse cholesterol transport activity. ABC1 fragments can have one or more amino acids deleted from any region of the polypeptide, including the carboxyl terminus and / or amino terminus, so long as biological function is maintained. ABC1 polypeptide fragments can occur naturally through alternative splicing or in vivo protease activity, or can be artificially constructed using well-known methods.

  The present invention also relates to ABC1 polypeptide derivatives which refer to chemically modified ABC1 polypeptides, variants or fragments as defined herein. Derivatives are modified to differ from a naturally occurring ABC1 polypeptide in either the type or position of the molecule attached to the polypeptide. Derivatives further include polypeptides formed by the deletion of one or more chemical groups naturally attached to the ABC1 polypeptide. In addition, ABC1 polypeptide comprising the amino acid sequence of SEQ ID NO: 2 and the ABC1 variants and fragments can be fused to homologous polypeptides to form homodimers, or fused to heterologous polypeptides to form heterodimers. Can be formed.

  Another aspect of the invention relates to mutant ABC1 polypeptides and fragments thereof corresponding to polypeptides isolated from Tangier patients. In one preferred embodiment, the ABC1 polypeptide comprises SEQ ID NO: 8. The protein is isolated from a Tangier patient (TD1) and sequenced as described in Examples 1 and 5. The amino acid sequence set forth in SEQ ID NO: 8 is similar to the wild type sequence with the exception of substitution of glutamine to arginine residue at position 537 (residue numbers are (Lawn et al., J. Clin. Invest., 104: r25-31 (1999)), which corresponds to position 597 of SEQ ID NO: 8. The position of this residue is in the amino-terminal hydrophilic domain and is the first predicted transmembrane Near the domain, the substitution alters the charge of amino acids in this region of the protein, resulting in an ABC1 protein with significantly reduced cholesterol efflux activity, as shown in FIG.

  In another preferred embodiment, the mutant ABC1 polypeptide comprises SEQ ID NO: 10. The protein corresponds to a polypeptide isolated from a Tangier patient (TD2) and is sequenced as described in Examples 1 and 5. The amino acid sequence set forth in SEQ ID NO: 10 is similar to the wild type sequence with the exception of substitution of arginine with tryptophan at residue 527 (residue number is Lawn et al., Supra (1999), which is SEQ ID NO: : Corresponding to position 587 of 10). Similar to the TED1 polypeptide, this substitution changes the charge of amino acid residues in the amino-terminal hydrophilic domain of the ABC1 protein. The resulting mutant ABC1 protein also has a significantly reduced cholesterol efflux activity, as shown in FIG.

(ABC1 polynucleotide)
Another aspect of the invention relates to isolated polynucleotides that encode the novel ABC1 polypeptides and variants thereof. The invention includes, for example, an isolated polynucleotide encoding a full-length ABC1 polypeptide, a polynucleotide containing the full-length cDNA of wild-type ABC1, a polynucleotide comprising the full-length coding sequence of wild-type ABC1, and a non-- Polynucleotides comprising coding 5 ′ and 3 ′ sequences are provided. The invention also provides an isolated polynucleotide encoding a mutant ABC1 polypeptide, such as that of a Tangier patient.

In one preferred embodiment, the isolated polynucleotide comprises a nucleotide sequence encoding a polypeptide comprising SEQ ID NO: 2. Importantly, in contrast to the published sequence of Langmann et al., Which codes for a protein of 2201 amino acids based on the predicted initiation methionine found in exon 3 (Langmann et al., Biochem, Biophys. Res. Comm. 257: 29-33 (1999) (GenBank accession number AJO12376), the currently claimed nucleotide sequence contains 50 exons and codes for a protein of 2261 amino acids (see FIG. 4). The nucleotide sequence consists of the following 60 amino acids-terminal amino acids:
MACWPQLRRLLLWKNLTFRRRQTCQLLLEVAWPLFIFFILISVRRLSYPPYEQHECHFPNKA
Contains a coding sequence comprising an additional 180 nucleotides at the 5 ′ end corresponding to.

  Assuming there are 6 to 9 nucleotides of in-frame stop codon upstream from this position, the newly predicted start site is a tax methionine codon that can produce a continuous open reading frame. The alignment of the related ABC transporter sequences ABCR and ABC-C (also known as ABC3), which also contains an open reading frame for the 60 additional amino acids, with this new ABC1 cDNA sequence shows a high degree of similarity; This means that the homologous ABC transporter protein begins with a sequence related to the amino terminal extension sequence proposed in human ABC1. The early published start site of human ABC1 is a published mouse ABC1 cDNA sequence containing an extra nucleotide “n” in the extension region so that the newly disclosed methionine is not in frame (Luciani et al., Genomics, 21150-159 (1994); GenBank accession number: X75926). However, if the “n” nucleotides in the mouse sequence are ignored, the mouse and human sequences in the extension region are identical. In view of these results, the full-length human ABC1 protein appears to contain 2261 amino acids rather than 2201 amino acids, as previously entered by Langmann et al. Thus, Langmann et al. Do not present a sufficient open reading frame for human ABC1.

  In another preferred embodiment, the isolated polynucleotide comprises a full length ABC1 cDNA comprising at least a portion of any of the non-coding 5 'and 3' sequences. Preferably, the polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 1. The 10.4 kb human ABC1 cDNA sequence shown in SEQ ID NO: 1 contains 6783 nucleotides + 5 'and an open reading frame of the 3' untranslated region. There is a start codon at position 291 and a stop codon at position 7074. The current ABC1 cDNA shown in SEQ ID NO: 1 differs from the published ABC1 cDNA (GenBank accession number. AJO12376) in several respects. First, the current ABC1 cDNA contains an additional 350 nucleotides at the 5 'end and an additional 3136 nucleotides at the 3' end (without the poly (A) tail). Also, the ABC1 sequence of the present invention differs from the ABC1 cDNA published by a 10 nucleotide substitution in the coding region. Of the 10 differences, 7 nucleotide differences predict amino acid changes. In order to be consistent with published records, the following nucleotide and amino acid numbers are shown in Lawn et al. , Clin. Invest. 104: R25-31 (1999) and GenBank accession number AJO12376. The nucleotide and amino acid changes are as follows: (1) A instead of G at nucleotide 414; (2) A instead of G at nucleotide 596 (K instead of R at amino acid 159); (3) Nucleotide 705 (4) A instead of C at nucleotide 1980; (5) A instead of G at 2413 (I instead of V at amino acid 765); (6) G instead of A at 2589 (M in place of I at amino acid 823); (7) T in place of C in 4604 (I in place of T in amino acid 1495); (8) T in place of C in 4883 (in place of P in amino acid 1588) L); (9) A instead of G in 5861 (of R in amino acid 1914 Spite of K); and (10) of C in 6443 instead of T (L instead of P in the amino acid 2108). Five of the amino acid changes are conservative amino acid changes and may represent polymorphisms or sequence errors. In two examples, the sequences of the invention predict significant amino acid differences from the GenBank sequence. The difference results in leucine rather than proline at residues 1588 and 2108. Interestingly, at the therapeutic site, the predicted leucine was also found in each of the three TD samples analyzed as well as the highly conserved mouse ABC1 protein.

  The invention also relates to an ABC1 polynucleotide comprising a nucleotide sequence that preferably has at least 80% identity over its entire length to a polynucleotide comprising SEQ ID NO: 1. More preferably, the polynucleotide has at least 90% identity over its entire length to the polynucleotide comprising SEQ ID NO: 1. Even more preferably, the polynucleotide has at least 95% identity over its entire length to the polynucleotide comprising SEQ ID NO: 1. Most preferably, the polynucleotide has 100% identity over its entire length to the polynucleotide comprising SEQ ID NO: 1. Such related ABC1 polynucleotides include substitutions, deletions and insertion variants, as well as allelic variants, splice variants, fragments, derivatives and homologous species, wherein one or more nucleotides are substitutions, deletions, insertions Or derivatized. Preferred polynucleotides include those that encode ABC1 polypeptides that possess biological activity such as cholesterol efflux activity.

  In another preferred embodiment, the isolated polynucleotide comprises the entire coding sequence of ABC1. In a particularly preferred embodiment, the polynucleotide exhibits the sequence set forth in nucleotides 291-7074 of SEQ ID NO: 1. This isolated polynucleotide contains a 6783 nucleotide ABC1 open reading frame and encodes a 2261 amino acid polypeptide as described above.

  In yet another preferred embodiment, the isolated polynucleotide comprises a nucleotide sequence encoding an ABC1 variant polypeptide. In particular, an isolated polynucleotide comprises a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 2. Also preferred is an isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 2. Accordingly, the present invention encodes said ABC1 polypeptides, including the described substitutions, deletions and insertion variants, as well as ABC1 allelic variants, splice variants, fragments, derivatives, fusion polypeptides and homologous species Including polynucleotides. A preferred polynucleotide is a polynucleotide that encodes a polypeptide that possesses the biological activity of ABC1. In particular, nucleotides encoding polypeptides that mediate reverse cholesterol transport are preferred. Also preferred are polynucleotides that encode polypeptides having improved reverse cholesterol transport activity.

  Yet another aspect of the invention relates to an isolated polynucleotide encoding a mutant ABC1 polypeptide from a Tangier patient. In one preferred embodiment, the polynucleotide encodes the polypeptide of SEQ ID NO: 8, which is isolated from patient TD1, which has been described above. In another preferred embodiment, the polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 7. The nucleotide sequence set forth in SEQ ID NO: 7 is sufficient to include an open reading frame, as well as 5 'and 3' flanking sequences. The open reading frame uses a numbering of a 2261 amino acid polypeptide (Lawn et al., Supra (1999)) containing a nucleotide substitution that results in an A to G substitution at position 537, among other substitutions. ).

  In another preferred embodiment related to a polynucleotide encoding a mutant ABC1 polypeptide, the polypeptide encodes the polypeptide of SEQ ID NO: 10, which polypeptide is isolated from Tangier patient TD2, which As mentioned above. In yet another preferred embodiment, the polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 9. The nucleotide sequence set forth in SEQ ID NO: 9 sufficiently comprises an open reading frame and 5 'and 3' flanking sequences. The open reading frame encodes a 2261 amino acid polypeptide containing a polynucleotide substitution that results in an Arg to Tryp substitution at residue 527, among other substitutions (using Lawn et al., Supra (1999) numbering). .

  Another aspect of the invention relates to an isolated polynucleotide comprising non-coding 5 'flanking and 3' flanking regions of ABC1. In one embodiment, the isolated polynucleotide comprises a non-coding 5 'flanking region of ABC1. Preferably, the 5 'flanking region includes, but is not limited to, the ABC1 promoter region. Thus, in a preferred embodiment, the polynucleotide comprises the sequence shown in SEQ ID NO: 3. As shown by the heterologous reporter assay and discussed in Example 15, the polynucleotide set forth in SEQ ID NO: 3 contains the transcriptional regulatory region of the ABC1 gene. As shown in FIG. 13, the polynucleotide set forth in SEQ ID NO: 3 contains several transcriptional regulatory elements, including TATA boxes at positions 1522, 1435 and 1383, as well as several putative SP1 sites and several nuclei. Containing a transcription factor binding site including a reporter half site 1643b. p. Non-coding sequence. In addition, the identified sterol response element is found at positions 1483-1500. Additional heterologous reporter assays described in Example 17 revealed that several distinct portions of SEQ ID NO: 3 possess promoter activity. Accordingly, in another preferred embodiment, the polynucleotide comprises nucleotides 1-1532, 1080-1643, 1181-1634, 1292-1643 or 1394-1643 of SEQ ID NO: 3. In a particularly preferred embodiment, the polynucleotide comprises nucleotides 1394-1532 of SEQ ID NO: 3, which was shown to have ABC1 promoter activity (see Example 17). In yet another preferred embodiment, the polynucleotide comprises nucleotides 1480-1510 of SEQ ID NO: 3, which is shown to modulate the ABC1 transcriptional response to the LXR ligand.

  The 5 'flanking polynucleotide also includes a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence set forth in SEQ ID NO: 3, wherein the nucleotide sequence has ABC1 promoter activity. In yet another embodiment, the polypeptide hybridizes under stringent conditions to a nucleotide sequence comprising nucleotides 1-1532, 1080-1643, 1181-1634, 1292-1643, or 1394-1643 of SEQ ID NO: 3. Wherein the nucleotide sequence has ABC1 promoter activity.

  In another embodiment, the isolated polynucleotide comprises the 3 'flanking region of ABC1. Several 3 'untranslated regions have been identified and entered that can represent alternative sites for polyadenylation of the ABC1 transcript. Preferably, the 3 'flanking region contains regulatory sequences. For example, the full length 3'UTR (SEQ ID NO: 6) contains 46 sequences (AA) nCU / UC (AA) n that have been shown to be required for binding of Vigilin. Vigilin, a universal protein with a 14K homology domain, is an estrogen-inducible vitellogenin mRNA 3'-untranslated region binding protein (J. Biol. Chem., 272: 12249-12252 (1997)). In addition to bound HDL, Vigilin has been shown to bind to the 3 ′ flanking region of mRNA and increase the half-life of the mRNA transcript (Mol. Cell. Biol., 18: 3991-4003 (1998)). . Thus, the 3'-flanking region could be altered, for example, to increase Vigilin binding, thereby increasing the half-life of ABC1 mRNA. Preferably, the isolated polynucleotide comprises the sequence set forth in SEQ ID NO: 4. Also preferably, the isolated polynucleotide comprises the sequence set forth in SEQ ID NO: 5. In another preferred embodiment, the isolated polynucleotide comprises the sequence shown in SEQ ID NO: 6. In other preferred embodiments, the polynucleotide comprises a sequence that hybridizes under stringent conditions to the nucleotide sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

  The present invention also includes related ABC1 5 'and 3' flanking polynucleotides. Accordingly, the present invention provides a polynucleotide comprising SEQ ID NO: 3, a polynucleotide comprising SEQ ID NO: 4, a polynucleotide comprising SEQ ID NO: 5, a polynucleotide comprising SEQ ID NO: 6, or nucleotide 1 of SEQ ID NO: 3. -1532, 1080-1643, 1181-1634, 1292-1643 or a polynucleotide comprising a nucleotide sequence having at least 80% identity over its entire length to the polynucleotide comprising. Preferably, the polynucleotide is at least 80%, more preferably at least 90%, more preferably at least 95%, most preferably 100% identity over its entire length to any one of the flanking polynucleotides described above. Have Preferred polynucleotides include polynucleotides that possess biological activity such as transcriptional regulatory activity.

  It is further understood that the present invention relates to an isolated polynucleotide that is complementary to any one of the aforementioned polynucleotide sequences. As used herein, the term “complementary” refers to hybridization or base pairing between nucleotides, for example, between two strands of a double-stranded polynucleotide. Two double-stranded nucleotide molecules are complementary when one strand of nucleotides is optimally aligned with the appropriate nucleotide insertion, deletion or substitution and paired with at least about 80% of the other strand of nucleotides. It is said that there is.

  Another aspect of the invention relates to a composition comprising the novel ABC1 described above and a suitable carrier. In one embodiment, the composition comprises a polynucleotide encoding a polypeptide comprising SEQ ID NO: 2, a polynucleotide comprising SEQ ID NO: 1, a polynucleotide comprising nucleotides 291-7074 of SEQ ID NO: 1, SEQ ID NO: A polynucleotide encoding a polypeptide comprising an amino acid sequence that is at least 98% identical to the amino acid sequence of 2, and a carrier. In another embodiment, the composition comprises a polynucleotide and a carrier that have at least 80%, preferably 90%, more preferably 95% identity over its entire length to the polynucleotide comprising SEQ ID NO: 1. .

  In another embodiment, the composition comprises ABC1 5 'flanking sequences and a suitable carrier. Preferably, the composition comprises a polynucleotide comprising SEQ ID NO: 3, or a polynucleotide comprising nucleotide fragments 1-1532, 1080-1643, 1181-1634, 1292-1643 or 1394-1643 of SEQ ID NO: 3 and suitable A suitable carrier. Also preferably, the composition comprises a polynucleotide having at least 80%, 90% or 95% identity over its entire length of a polynucleotide comprising any one of the 5 ′ flanking sequences described and a suitable carrier. Including. In yet another embodiment, the composition comprises a polynucleotide comprising ABC1 3 'flanking sequences and a suitable carrier. A preferred composition comprises a polynucleotide comprising SEQ ID NO: 4, a polynucleotide comprising SEQ ID NO: 5, or a polynucleotide comprising SEQ ID NO: 6, and at least 80% over its entire length relative to any of these polynucleotides Preferably 90%, more preferably 95% identity polynucleotide and a suitable carrier. Yet another composition of the invention comprises a mutant ABC1 polynucleotide. Preferably, the composition comprises a polynucleotide comprising SEQ ID NO: 7 or a polynucleotide comprising SEQ ID NO: 9 and a suitable carrier.

In addition, the compositions of the invention comprise two or more of the ABC1 polynucleotides and a suitable carrier in any combination. Any suitable aqueous carrier can be used in the composition. Preferably, the carrier stabilizes the composition at the desired temperature such as room temperature or storage temperature (ie, 4 ° C. to 20 ° C.) and is of a suitable neutral pH. Examples of suitable carriers are known to those skilled in the art, including tris -EDTA and DEPC-H ₂ O.

(ABC1 vector and host cell)
The present invention also relates to a recombinant vector comprising one or more ABC1 polynucleotides, host cells genetically engineered with the vector comprising ABC1 polynucleotides, and production of ABC1 polynucleotides by recombinant techniques. As described above, the present invention provides a recombinant vector comprising one or more wild-type ABC1 polynucleotides as described above. In a preferred embodiment, the recombinant vector comprises a polynucleotide encoding a polypeptide comprising SEQ ID NO: 2, a polynucleotide comprising SEQ ID NO: 1, and a polynucleotide comprising nucleotides 291-7074 of SEQ ID NO: 1. In another preferred embodiment, the recombinant vector comprises a variant polynucleotide encoding a polypeptide comprising an amino acid sequence that is at least 98% identical to the amino acid of SEQ ID NO: 2. In another preferred embodiment, the recombinant vector comprises a variant polynucleotide that is at least 80% identical to the polynucleotide comprising SEQ ID NO: 1. In yet another preferred embodiment, the recombinant vector comprises a polynucleotide that is complementary to any of these polynucleotides.

  In another embodiment, the recombinant vector comprises a polynucleotide comprising a mutant ABC1 polynucleotide isolated from a Tangier disease patient. In a preferred embodiment, the recombinant vector comprises a polynucleotide comprising SEQ ID NO: 8. In another preferred embodiment, the recombinant vector comprises a polynucleotide comprising SEQ ID NO: 10. Recombinant vectors also contain polynucleotide sequences that are complementary to these sequences.

  It is also understood that the recombinant vector can include two or more of the wild type, variant, or mutant ABC1 polynucleotides in any combination.

  An isolated ABC1 polynucleotide, such as any of the wild-type, variant, or mutant polynucleotides described above, is inserted into the vector using well-known ligation and cloning techniques. Cloning techniques are described in Davis et al., Basic Methods in Molecular Biology (1986); Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, (Cold Spring Harbor Press, Cold N. Bold A. Ed., Current Protocols in Molecular Biology (1991), (Wiley and Sons (1994)); Goeddel ed., Gene Expression Technology (Methods in Enzymology (Trade in 1991); n Protocols are described in several standard manuals, including (Human Press, Clifton, NJ) to.

  Any vector suitable for ABC1 polynucleotide insertion can be used. The vector is typically selected to function in the particular host cell used (ie, the vector is compatible with the host cell machine such that gene amplification and / or expression can occur). Preferably, the vector is compatible with a bacterial, insect or mammalian host cell. Also preferably, the vector is an expression vector (for reviews of expression vectors, see Goeddel, DV, Ed., Methods Enzymol., Academic Press Inc., San Diego, CA (1990)). The vector can be, for example, a phage, plasmid, virus, or retroviral vector. Retroviral vectors can be replication competent or replication defective. In the latter case, viral propagation will generally occur only in supplemented host cells.

  Typically, expression vectors used in any of the host cells contain sequences for plasmid maintenance and for the expression of exogenous nucleotide sequences. Such a sequence collectively referred to as a “flanking sequence” is preferably a complete intron containing the following nucleotide sequence: promoter, one or more enhancer sequences, origin of replication, transcription termination sequence, donor and acceptor splice sites One of a sequence, a sequence encoding a leader sequence for polypeptide insertion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting a nucleic acid encoding a polypeptide to be expressed, and a selectable marker element Should include the above.

  Flanking sequences can be homologous (ie, the same species and / or strain as the host cell), heterologous (ie, species other than the host cell species or strain), hybrid (ie, combinations of flanking sequences from more than one source), Or it can be synthetic. A flanking sequence may also be a native sequence that normally functions to regulate ABC1 polypeptide expression. The source of the flanking sequence can be prokaryotic or eukaryotic, any vertebrate or invertebrate, or any plant, but the flanking sequence can function in and be activated by the host cell machine Shall.

  The vector should also preferably include at least one selectable marker for propagation in the host. Selectable markers are genetic elements that encode proteins necessary for the survival and growth of host cells that grow on selective media. Appropriate selectable marker genes confer resistance to antibiotics or other toxins for prokaryotic host cells, such as ampicillin, tetracycline, or kanamycin; Or (c) encodes a protein that supplies critical nutrients that are not available from the complex medium. Preferred selectable markers are zeocin, G418, hygromycin or neomycin resistance and E. coli for eukaryotic cultures. including tetracycline, kanamycin, or ampicillin resistance for culturing E. coli and other bacteria.

  Other suitable selection genes include those used to amplify the expressed gene. Amplification is the process by which genes that are in great demand for the production of proteins critical for growth repeat in tandem within the chromosomes of successive generations of recombinant cells. Examples of suitable selectable markers for mammalian cells include dihydrofolate reductase (DHFR) and thymidine kinase. Mammalian cell transformants are placed under selective pressure, where only the transformants are uniquely adapted to survive with the selection gene present in the vector. Selection pressure is imposed by culturing the transformed cells under conditions where the concentration of the selective agent in the medium changes continuously, thereby leading to amplification of both the selection gene and the ABC1 gene. As a result, increased amounts of ABC1 polypeptide are synthesized from the amplified DNA.

  The vector also preferably contains a transcription termination sequence that is typically located 3 'to the end of the polypeptide coding sequence and serves to terminate transcription. Usually, the transcription termination sequence in prokaryotes is a GC rich fragment followed by a poly T sequence. The sequence is purchased as part of a commercial vector or synthesized using well-known methods for nucleic acid synthesis.

  The vector also preferably contains a ribosome binding site that is usually required for translation initiation of mRNA and is characterized by a Shine-Dalgarno sequence (prokaryotes) or a Kozak sequence (eukaryotes). The element is typically 3 'to the promoter and 5' to the coding sequence of the ABC1 polypeptide to be expressed. Shine-Dalgarno sequences have been identified, each of which can be readily synthesized by well-known methods.

  The vector should also preferably include a promoter that is recognized by the host organism and operably linked to the encoded polynucleotide. The promoter can be an inducible promoter or a constitutive promoter. Inducible promoters initiate transcription from increased levels of DNA under their control in response to certain changes in culture conditions, such as the presence or absence of nutrients, or changes in temperature. In contrast, constitutive promoters initiate continuous gene product production; as a result, there is little or no control over gene expression. A suitable promoter is operably linked to the polynucleotide by removing the promoter from the source DNA by restriction enzyme digestion and inserting the desired promoter sequence into the vector. As described above, a natural promoter can be used to direct amplification and / or expression of a polynucleotide. Thus, the recombinant vector may include one of the wild type, variant, or mutant ABC1 polynucleotides and ABC1 promoter described above, such as those found in SEQ ID NO: 3. A recombinant vector can also include two or more of the wild type, variant or mutant ABC1 polynucleotides and ABC1 promoter in any combination.

  Preferably, if it gives greater transcription and higher yield of ABC1 protein compared to the native ABC1 promoter, and if it is compatible with the host cell system selected for use, it is heterologous. Use a promoter. Heterologous promoters can be used alone or in combination with the native ABC1 promoter. Thus, in one preferred embodiment, a recombinant vector comprises any one of said wild type ABC1 polynucleotide and a heterologous promoter. In another preferred embodiment, the recombinant vector comprises any one of said variant ABC1 polynucleotide and a heterologous promoter. In yet another preferred embodiment, a recombinant vector comprises any one of the mutant ABC1 polynucleotide and a heterologous promoter. Preferred embodiments also include a recombinant vector comprising any combination of two or more of the wild type, variant and mutant ABC1 polynucleotides and a heterologous promoter.

  Suitable heterologous promoters for use in prokaryotic hosts include, but are not limited to, beta-lactamase and lactose promoter systems (Villa-Kamaroff et al., 1978, Proc. Naltl. Acad. Sci. USA, 75: 3727-31), alkaline phosphatase, tryptophan (trt) promoter system, and tac promoter (Villa-Kamaroff et al., 1978, Proc. Naltl. Acad. Sci. USA, 75: 3727-31). Includes hybrid promoters. Their sequences have been published, thereby allowing those skilled in the art to link them to the desired DNA sequence using the linkers or adapters necessary to provide any useful restriction sites. Other suitable heterologous promoters are known to those skilled in the art.

  Heterologous promoters suitable for use in mammalian host cells are well known and include, but are not limited to, polyoma virus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, birds , Sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus and simian virus 40 (SV40). Other suitable mammalian promoters include heterologous mammalian promoters, such as heat-shock promoters and actin promoters. Further suitable promoters include, but are not limited to, the SV40 early promoter and the promoter region described below (Bernoist and Chamber, 1981, Nature 290: 340-10); the promoter included in the 3 'long terminal repeat of Rous sarcoma virus ( Yamamoto et al., 1980, Cell 22: 787-97); herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. S. A. 78: 1444-45); and regulatory sequences of the metallothionine gene ( Brinster et al., 1982, Nature 296: 39-42). Preferably, the promoter is a cytomegalovirus or SV40 promoter. Thus, in a particularly preferred embodiment, the recombinant vector comprises one of said wild type ABC1 polynucleotide, one of said variant ABC1 polynucleotide, or of said mutant ABC1 polynucleotide and cytomegalovirus promoter. One of these. In another particularly preferred embodiment, the recombinant vector comprises two or more of the wild type, variant, or ABC1 polynucleotide and cytomegalovirus promoter in any combination.

  The vector also preferably includes an enhancer sequence for increasing transcription of the polynucleotide, such as ABC1. Suitable enhancers for activation of eukaryotic promoters include viral enhancers such as SV40, cytomegalovirus early promoter, polyoma and adenovirus enhancers.

  The expression vectors of the invention can be constructed from starting vectors such as commercially available vectors that may or may not contain all of the desired flanking sequences. If one or more of the flanking sequences described herein are not already present in the vector, they can be obtained individually and linked to the vector. The methods used to obtain each of the flanking sequences are well known to those skilled in the art.

  Preferred vectors are those that are compatible with bacterial, insect and mammalian host cells. Preferred vectors for use in bacteria include, for example, pQE70, pQE60 and pQE-9 (Quiagen. Inc.) blue script vectors, phage script vectors, pNH16A, pNH18A, pNH46A (Stratagene Cloning Systems, Inc.), prc99a, pKK223-. 3, pDR540, pRIT5 (Pharmacia Biotech, Inc.), and pCEP4 (Invitrogen Corp., Carlsbad, CA). Preferred eukaryotic vectors include, but are not limited to, pWLNEO, pSV2CAT, pOG44, pXTI and pSG ((Stratagene), pSVK3, pBPV, pMSG and pSVL (Pharmacia) and pGL3 (Promega, Medison, WI). Other suitable vectors will be readily apparent to those skilled in the art.

  In a particularly preferred embodiment, the recombinant vector contains pCEPhABC1 described in Example 4 and shown in FIG. Recombinant vector pCEPhABC1 includes plasmid pCEP4 (an expression vector containing Invitrogen, cytomegalovirus promoter and enhancer. Vector pCEPhABC1 further includes an ABC1 polynucleotide operably linked to a heterologous cytomegalovirus promoter. ABC1 polynucleotide Contains SEQ ID NO: 1 containing the full-length ABC1 cDNA including the non-coding 5 ′ flanking (ie, the native ABC1 promoter) and the 3 ′ flanking sequence.

  In addition, the present invention provides a recombinant vector comprising an ABC1 flanking sequence polynucleotide. In one embodiment, the recombinant vector comprises a polynucleotide comprising an ABC1 5 'flanking sequence, preferably containing promoter activity. Thus, in a preferred embodiment, the recombinant vector comprises a polynucleotide comprising SEQ ID NO: 3. In another preferred embodiment, the recombinant vector comprises a polynucleotide comprising nucleotides 1-1532, 1080-1643, 1181-1634, 1292-1643, or 1394-1643 of SEQ ID NO: 3. In a particularly preferred embodiment, the polynucleotide comprises nucleotides 1394-1532 of SEQ ID NO: 3. In another embodiment, the recombinant vector is a polynucleotide set forth in SEQ ID NO: 3 or nucleotides 1-1532, 1080-1643, 1181-1634, 1292 of SEQ ID NO: 3 under stringent conditions. 1643, or a polynucleotide that hybridizes to a polynucleotide comprising 1394-1643. In yet another embodiment, the polynucleotide comprises a polynucleotide comprising SEQ ID NO: 3 or a polynucleotide comprising nucleotides 1-1532, 1080-1643, 1181-11643, 1292-1643, or 1394-1643 of SEQ ID NO: 3. A nucleotide sequence having at least 80%, more preferably at least 90%, and even more preferably at least 95% identity over its entire length to the nucleotide. The recombinant vector also includes a polynucleotide complementary to any of the 5 'flanking sequences.

  In another embodiment, the recombinant vector comprises a polynucleotide comprising the ABC1 3 'flanking sequence. In a preferred embodiment, the recombinant vector comprises a polynucleotide comprising SEQ ID NO: 4. In another preferred embodiment, the recombinant vector comprises a polynucleotide comprising SEQ ID NO: 5. In an equally preferred embodiment, the recombinant vector comprises a polynucleotide comprising SEQ ID NO: 6. In another embodiment, the recombinant vector comprises a polynucleotide that hybridizes under stringent conditions to the polynucleotide set forth in SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. In yet another embodiment, the polynucleotide is at least 80%, more preferably at least 90%, even more preferably over its entire length relative to a polynucleotide comprising SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 Comprises a nucleotide sequence having at least 95% identity. The recombinant vector also includes a polynucleotide that is complementary to any of the 3 'flanking sequences.

  An isolated ABC1 flanking sequence polynucleotide, such as either the 5 'or 3' flanking sequence polynucleotide, is inserted into the vector using well-known ligation and cloning techniques. Any of the vectors described above can be used. Preferably, the vector is compatible with bacterial, insect or mammalian host cells. Also preferably, the vector is an expression vector. The vector can be, for example, a phage, plasmid, virus or retroviral vector.

  In addition to the ABC1 flanking sequence, the vector includes the following flanking nucleotide sequences: promoter, one or more enhancer sequences, origin of replication, transcription termination sequence, complete intron sequence containing donor and acceptor splice sites, polypeptide secretion Containing one or more of a sequence encoding a leader sequence, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting a nucleic acid encoding a polypeptide to be expressed, and a selectable marker element it can. Any of the flanking nucleotide sequences described above are suitable. The flanking sequences can be homologous, heterologous, hybrid or synthetic. A flanking sequence can also be a native sequence that normally functions to regulate ABC1 polypeptide expression. The source of the flanking sequence can be prokaryotic or eukaryotic, either vertebrate or invertebrate, or any plant, but the flanking sequence functions in the host cell machine and can be activated thereby And

  Preferred vectors are those that are compatible with bacterial, insect and mammalian host cells. Suitable vectors are those described above, pQE70, pQE60, pQE9, bluescript vectors, phage script vectors, pNH16A, pNH18A, pNH46A, ptrc99a, pkk223-3, pDR540, pRIT5 and pCEP4 used in bacteria, and eukaryotic cells. PWLNEO, pSV2CAT. pOG44, pXTI, pSG, pSVK3, pBPV, pMSG, pSVL and pGL3.

  In one particularly preferred embodiment, the recombinant vector comprises a polynucleotide comprising the 5 'flanking region of the ABC1 gene and further comprises at least one polynucleotide encoding a heterologous polypeptide. The heterologous polynucleotide is operably linked to the ABC1 5 'flanking sequence. The ABC1 5 'flanking sequence preferably contains the ABC1 promoter. Thus, preferably the 5 'flanking sequence comprises the sequence set forth in SEQ ID NO: 3. Equally preferably, the 5 'flanking sequence comprises nucleotides 1-1532, 1080-1643, 1181-1634, 1292-1643 or 1394-1643 of SEQ ID NO: 3. The heterologous polynucleotide preferably encodes a polypeptide that is a complete protein or a biologically active fragment of a protein. A vector can contain more than one heterologous polynucleotide. More preferably, the heterologous polynucleotide encodes a reporter protein. In such cases, the recombinant vector preferably does not contain any additional promoter sequences. Examples of suitable reporter proteins include luciferase, β-galactosidase, chloramphenicol acetyltransferase transferase, and green fluorescent protein. Preferably, the reporter polynucleotide is luciferase. Thus, in one particularly preferred embodiment, the recombinant vector comprises the 5 'flanking sequence set forth in SEQ ID NO: 3 and a luciferase reporter polynucleotide. In other equally preferred embodiments, the recombinant vector comprises a polynucleotide comprising nucleotides 1-1532, 1080-1643, 1181-1634, 1292-1643 or 1394-1643 of nucleotide SEQ ID NO: 3 and a luciferase reporter polynucleotide. Including.

  An expression vector comprising an ABC1 5 'flanking sequence can be constructed from a starting vector such as a commercially available vector containing a reporter polynucleotide. Examples of suitable expression vectors include pGL3-Basic containing a luciferase reporter gene (Promega, Madison, WI and pβGal-Basic (Clontech, Palo Alto, Calif.) A preferred vector is a promoterless pGL3-Basic luciferase reporter vector. A 5 ′ flanking sequence containing the ABC1 promoter, eg, SEQ ID NO: 3, can be generated using well-known methods, including those described herein (see Example 15). Thus, in a particularly preferred embodiment, the recombinant vector is a reporter gene construct comprising pAPR1, SEQ ID NO: 3 and a luciferase reporter gene in a pGL3 vector. Yes (see FIG. 11).

  The present invention also relates to a host cell containing any one of the above recombinant vectors. After the vector is constructed, the completed vector can be inserted into a suitable host cell for amplification and / or polypeptide expression. The host cell can be a prokaryotic host cell (such as E. coli) or a eukaryotic host cell (such as a yeast cell, insect cell or vertebrate cell). When cultured under appropriate conditions, the host cell synthesizes an ABC1 polypeptide or a reporter polypeptide that can subsequently be measured. The host cell can be a mammalian host cell such as a primate cell line, including a transformed cell line, or a rodent cell line. Normal diploid cells, cell lines derived from in vitro culture of primary tissues, and primary explants are also suitable. Candidate host cells can be phenotypically defective in the selection gene or can predominantly contain the action selection gene.

  A number of suitable host cells are known in the art and are available from the American Type Culture Collection (ATCC), Manassas, VA. Suitable mammalian host cells include, but are not limited to, Chinese hamster ovary cells (CHO), CO DHFR-cells (Urlaub et al., 1980, Proc. Natl. Acad. Sci., 97: 4216: 20), Human embryonic kidney (hek) 293 or 293T cells, or 3T cells, monkey COS-1 and COS-7 cell lines, and CV-1 cell lines. Other suitable mammalian cell lines include, but are not limited to, mouse neuroblastoma N2A cells, HeLa cells, mouse 1-929 cells, Swiss-derived 3T3 lines, Balb-c or NIH mice, BHK or HAK hamster cell lines, Thp-1, HepG2 and mouse RAW cell lines. Each of these cell lines is known and available to those skilled in the art of protein expression. A preferred host cell is the murine monocytic cell line RAW 264.7 whose use is described in Example 8.

  Suitable bacterial host cells are those that are well known as host cells in the field of biotechnology. various strains of E. coli (eg, HB101, DH50, DH10, and MC1061). B. subtilis, Pseudomonas spp. , Other Bacillus spp. , Streptomyces spp. Various strains such as can also be used in this method. Many strains of yeast cells are also available as host cells for expression of ABC1 polypeptides. Preferred yeast cells include, for example, Saccharomyces cerevisiae and Pichia pastoris. In addition, insect cell lines can be suitable host cells. Insect cell lines are described, for example, by Kitts et al., 1993, Biotechniques, 14: 810-17; Lucluck, 1993, Curr. Opin. Biotechnol. 4: 564-72; and Luclow et al., 1993, J. MoI. Virol. 67: 4566-79. Preferred insect cells are Sf-9 and Hi5 (Invitrogen).

  The invention also includes (a) transfecting a mammalian host cell with a recombinant expression vector comprising a polynucleotide encoding ABC1 sufficient to produce a detectable level of ABC1 protein, and then (b ) A method for expressing ABC1 protein in a mammalian host cell comprising the step of purifying the resulting ABC1 protein. In one preferred embodiment, the recombinant expression vector comprises a polynucleotide encoding a polypeptide comprising SEQ ID NO: 2. In another preferred embodiment, the polynucleotide comprising SEQ ID NO: 1. In yet another preferred embodiment, a polynucleotide comprising nucleotides 291-7074 of SEQ ID NO: 1. In yet another preferred embodiment, a polynucleotide encoding a polypeptide that is at least 98% identical to the polypeptide comprising SEQ ID NO: 2.

  Introduction of recombinant ABC1 vectors into mammalian host cells can be accomplished by methods well known in the art and are described in standard laboratory manuals such as Sambrook, supra. Preferably, the recombinant vector is introduced into the host cell in a precipitate or complex with a charged lipid. Suitable methods for introducing ABC1 vectors include calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods known in the art. These methods are described, for example, in Davis et al., Basic Methods in Molecular Biology (1986). If the recombinant ABC1 vector is a viral vector, it can be introduced into package C and then into the host cell in vitro using an appropriate packaging cell line.

  ABC1-polypeptides are well-suited to include ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lecithin chromatography. It can be recovered from recombinant cell culture and purified using known methods [see, eg, Smith and Johnson, Gene 63: 31-40 (1988)]. Preferably, affinity chromatography using anti-ABC1 antibody is used for purification.

  In addition, the present invention includes the step of administering to a mammalian subject an expression vector comprising a sufficient amount of a polynucleotide encoding ABC1 to express the ABC1 protein in the mammalian subject. Provides a method for expressing the ABC1 protein. Preferably, the recombinant expression vector is a polynucleotide encoding a polypeptide comprising SEQ ID NO: 2, a polynucleotide comprising SEQ ID NO: 1, a polynucleotide comprising nucleotides 291-7074 of SEQ ID NO: 1, or SEQ ID NO: 2 A polynucleotide encoding a polypeptide that is at least 98% identical to the polypeptide comprising. Introduction of a recombinant ABC1 vector into a mammalian subject can be accomplished by methods well known in the art, which are described in detail herein below. ABC1 expression can be measured by obtaining a blood sample from a subject to which a recombinant ABC1 vector has been administered, separating a monocyte population, and measuring ABC1 gene expression in macrophage cells. ABC1 gene expression is a method well known in the art, such as RT-PCR, and the method described herein (see Examples 9 and 10). ABC1 protein levels are measured by obtaining a blood sample from a subject, separating a monocyte population, and measuring ABC1 protein in macrophage cells using well-known methods such as immunoprecipitation as described in Example 11. can do.

(Methods and compounds for increasing cholesterol efflux)
In another aspect of the invention, a method is provided that is suitable for increasing cholesterol efflux from cells in a mammalian subject. Such a method comprises administering to a mammalian control a recombinant expression vector comprising an ABC1 polynucleotide in an amount sufficient to increase nocholesterol efflux from the cell. A recombinant vector can be any of the vectors containing either the wild type or variant ABC1 polynucleotides described above so long as the encoded ABC1 polypeptide has biological activity (ie, cholesterol transport activity). . Preferably, the recombinant vector is a polynucleotide encoding a polypeptide comprising SEQ ID NO: 2, a polynucleotide comprising nucleotides 291-7074 of SEQ ID NO: 1, or at least 98% identical to the amino acid sequence of SEQ ID NO: 2. A polynucleotide encoding a polypeptide comprising an amino acid sequence which is Also preferably, the recombinant vector is a variant polynucleotide that is at least 80%, 90% or 95% identical to the polynucleotide comprising SEQ ID NO: 1 as long as the encoded ABC1 polypeptide has cholesterol transport activity including.

  Administration of a recombinant ABC1 expression vector to a mammalian subject can be used to express the ABC1 gene in the subject for the treatment of cardiovascular disease. Specifically, this method will achieve a therapeutic effect by introducing the ABC1 gene into macrophage cells and other cholesterol-accumulating cells found in arterial lesions of mammals with cardiovascular disease. Expression of ABC1 polynucleotides in target cells will result in greater production of ABC1 protein. Subsequent produced ABC1 protein will alleviate the disease by increasing the efflux of cholesterol from macrophages and other cholesterol-loaded cells found in arterial lesions to naked HDL particles. Cell efflux will lead to the removal of total cholesterol from peripheral sites such as the cholesterol-rich core of the arterial plaque. Simultaneous reduction in the size of these pathological lesions reduces the risk of arterial occlusion leading to heart attacks and angina. This method could also be used prophylactically to prevent cholesterol accumulation in the arterial wall.

  A sufficient amount of ABC1 expression vector is an amount of ABC1 vector that increases cholesterol efflux from cells of a mammalian subject. Such an amount measures cholesterol efflux in control cells before (control levels) and after administration of various doses of recombinant ABC1 expression vector and measures the dose that results in increased cholesterol efflux compared to control levels. Can be determined. Cholesterol efflux can be measured by obtaining a blood sample from the subject, separating the monocyte population, and measuring the amount of cholesterol efflux in the macrophage cells of the subject. Cholesterol efflux can be measured using any of the assays described herein.

  In addition, cholesterol efflux can be measured by determining the level of plasma HDL-cholesterol in the subject before (control) and after administration of the recombinant ABC1 expression vector. The observed increase in HDL-cholesterol in the subject's serum following administration of the recombinant ABC1 expression vector indicates an increase in cholesterol efflux. HDL-cholesterol in serum can be measured using methods well known in the art.

  In addition, cholesterol efflux can be measured by measuring the size of atherosclerotic lesions found in the control arterial wall before (control levels) and after administration of the recombinant ABC1 expression vector. A decrease in the size of the arterial lesion indicates an increase in cholesterol efflux. Assays for measuring arterial lesions are well known in the art. For example, increased cholesterol efflux from arterial lesions uses the mouse model of atherosclerosis described in Lawn et al., Nature, 360: 670-672 (1992), as well as any other known method. Can be measured. Using LDL receptor knockout mice described in Lawn et al., Cholesterol efflux can be measured before and after administration of ABC1 vector. The size of a fat streak lesion in a group of animals given an atherogenic diet can be measured by oil-red O staining of aortic sections described in Lawn et al. A decrease in the size of the fat streak lesion in the group receiving the ABC1 expression vector compared to the group receiving the control vector indicates an increase in cholesterol efflux from the lesion. In humans, the size of atherosclerotic lesions found in the arterial wall can be measured using, for example, angiography and noninvasive ultrasound methods.

  Alternatively, cholesterol efflux can be measured by obtaining a blood sample and then measuring the level of ABC1 mRNA or ABC1 protein in the subject macrophage cells before and after administration of the recombinant ABC1 expression vector. Routine assays can be performed to determine the correlation between ABC1 mRNA concentration and cholesterol efflux. Similarly, an assay can be performed to correlate the amount of ABC1 protein with the amount of cholesterol efflux. Such correlation data can be used to indicate an increase in cholesterol efflux using the observed increase in ABC1 mRNA or ABC1 protein in the cells of interest after administration of the recombinant ABC1 expression vector. ABC1 mRNA and ABC protein levels can be measured using the assays described herein and other well-known techniques for mRNA and protein quantification. The therapeutic dosage and formulation are discussed in detail later.

A number of viral and non-viral methods suitable for introducing nucleic acid molecules into target cells are available to those skilled in the art. For example, viral delivery vectors suitable for gene therapy include, but are not limited to, adenovirus, herpes simplex virus, poxvirus (ie, vaccinia), hepatitis virus, parvovirus, papovavirus, alphavirus, coronavirus, rhabdo Viruses, papillomaviruses, adeno-associated viruses (AAV), polioviruses, and RNA viruses such as retroviruses and Sindbis viruses. ABC1 polynucleotides can be used for naked DNA delivery (direct injection, receptor-mediated introduction (DNA-ligand complex), electroporation, adenovirus-ligand-DNA complex, calcium phosphate (CaPO ₄ ) precipitation, macroparticle bombardment ( It can be delivered using non-viral delivery systems such as gene gun technology), liposome-mediated transfer, and lipofection.

  Genetic modification of cells can be accomplished using one or more techniques well known in the field of gene therapy (Mulligan, R., 1993, Science, 260 (5110): 926-32). Gene therapy materials and methods include inducible promoters, tissue-specific enhancer-promoters, DNA sequences designed for site-specific integration, DNA sequences that can provide superior selection advantages over parental cells, A label to identify transformed cells, a negative selection system and an expression control system (a measure of safety), a cell-specific binding agent (for cell targeting), a cell-specific internalization factor, And transcription factors to enhance expression by the vector and methods of vector production. Examples of methods and materials for the practice of gene therapy techniques include US Pat. No. 4,970,154 (including electroporation techniques), No. 5 (describes lipoprotein-containing systems for gene delivery). 679, 559, 5,676, 954 (including liposome carriers), 5,593, 875 (which describes a method for calcium phosphate transfection), and biologically active particles 4,945,050, and PCT (including nuclear ligands), which describe the process by which cells are propelled at a certain speed, thereby allowing the particles to penetrate the surface of the cells and become integrated inside the cells. It is described in the publication number WO96 / 40958.

  Adenoviral vectors have been found to be particularly useful for gene transfer into eukaryotic cells (Rosenfeld, M. et al., Science, 252: 431-4 (1991); US Pat. No. 5,631,236). issue). The first trial of Ad-mediated gene therapy in humans was the introduction of the cystic fibrosis transmembrane conductance regulator (CFTR) gene into the lung (Crystal, R. et al., 1994, Nat. Genet., 8 (1). : 42-51). Experimental routes for administering recombinant Ad to different tissues in vivo are intra-organ instillation (Rosenfeld, M. et al., 1992, Cell, 68 (1): 143-55), intramuscular injection (Quantin. B., et al., Proc. Natl. Acad. Sci. USA, 89 (7): 2581-4), peripheral intravenous injection (Herz, J. and Gerard, R., 1993, Proc. Natl. Acad.Sci.U.S.A., 90 (7): 2812-6) and stereotaxic inoculation into the brain (Le Gal La La Sale, G., et al., 1993, Science, 259 (5097): 988-90. ). Accordingly, adenoviral vectors are widely available to those skilled in the art and are suitable for use in the present invention.

  Adeno-associated virus (AAV) has recently been introduced as a gene transfer system with excellent application in gene therapy. Wild-type AAV exhibits a high level of infectivity, broad host range and specificity in integrating into the host cell genome (Hermonat. P., and Muzyczka, N., 1984, Proc. Natl. Acad. Sci. U. SA, 81 (29): 6466-70). Herpes simplex virus type-1 (HSV-1) is a preferred vector system (Geller, A., et al., 1991, Trends Neurosci., 14 (10): 428-32; Glorioso, J., et al., 1995, Mol. Biotechnol., 4 (1): 87-99; Glorioso, J., et al., 1995, Annu. Rev. Microbiol., 49: 675-710). Vaccinia viruses from the Poxviridae family have also been developed as expression vectors (Smith, G., and Moss, B., 1983, Gene, 25 (1): 21-8; Moss, B., 1992, Biotechnology, 20 : 345-62; Moss, B., 1992, Curr. Top. Microbiol. Immunol., 158: 25-38). Each of the vectors is widely available to those skilled in the art and will be suitable for use in the present invention.

  Preferably, the viral delivery system utilizes a retroviral vector. Retroviral vectors can infect a large proportion of target cells and integrate into the cell's genome (Miller, A., and Rosman, G., Biotechniques, 7 (9): 980-2, 984-6, 989. -90 (1989); U.S. Pat. No. 5,672,510). Retroviruses were developed as gene transfer vectors relatively early than other viruses and were first and successfully used for gene marking and introduction of adenosine deaminase (ADA) cDNA into human lymphocytes. Preferably, the retroviral vector is a derivative of a murine or avian retrovirus or is a lentiviral vector. Particularly preferred retroviral vectors are lentiviral vectors. Examples of retroviral vectors that can introduce a single foreign gene include, but are not limited to, Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), SIV, BIV, HIV and Rous sarcoma virus RSV). A number of additional retroviral vectors can be incorporated into multiple genes. All of these vectors can be introduced or integrated into a gene for a selectable marker so that the introduced cell can be identified and regenerated.

  Since recombinant retroviruses are defective, it needs help to generate infectious vector particles. This help can be provided, for example, by using a helper cell line containing a plasmid encoding all of the retroviral structural genes under the control of regulatory sequences within the LTR. The helper plasmid lacks a nucleotide sequence that allows the packaging mechanism to recognize the RNA transcript for encapsulation. Thus, since the genome is not packaged, the helper cell line produces empty virions. Suitable helper cell lines include, but are not limited to ψ2, PA317 and PA12. If a retroviral vector is introduced into such a cell where the packaging signal is intact but the structural gene has been replaced by another gene of interest, the vector can be packaged to generate a vector virion. it can. The vector virions generated by this method can then be used to infect tissue cell lines such as NAIH 3T3 cells to produce large quantities of chimeric retroviral virions.

  The vectors of the present invention can be constructed using standard recombinant techniques widely available to those skilled in the art. Such techniques are described in Molecular Cloning: A Laboratory Manual (Sambrook et al., 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology, 19th. , CA), and PCR Protocols: A Guide to Methods and Applications (Innis et al., 1990, Academic Press, San Diego, Calif.).

  In order to obtain transcription of ABC1 polynucleotides in the target cell, a transcriptional regulatory region that can drive gene expression in the target cell must be utilized. The transcriptional regulatory region preferably includes a promoter and / or enhancer operably linked to the ABC1 polynucleotide. The promoter can be homologous or heterologous to the ABC1 gene provided that it is active in the cell or tissue-type into which the construct will be inserted. The selected transcriptional regulatory region should drive high levels of gene expression in the target cell. Preferably, a macrophage-specific promoter such as the scavenger receptor type A, matrix metalloproteinase promoter (MMP-12), or macrophage tropic lentivirus promoter (Fabuni et al., Atherosclerosis, 148: 375-386 (1999)) is used. . A particularly preferred promoter is the 5 'region of the scavenger receptor type A gene, which contains a strong macrophage promoter that can be used to drive transcription of the ABC1 gene. In addition, a means for increasing the expression of endogenous ABC1 polypeptide in the cell is to insert one or more enhancer elements in the promoter region, where the enhancer element appears to increase the transcriptional activity of the ABC1 gene. Can work. Similarly, the enhancer element used is selected based on the tissue in which the gene is desired to be activated. Thus, for example, an enhancer element known to confer promoter activation in cells found in arterial tissue, particularly macrophage cells, would be selected. Other transcriptional regulatory regions suitable for use in the present invention include, but are not limited to, human cytomegalovirus (CMV) immediate-early enhancer (promoter, SV40 early enhancer / promoter, JC polyomavirus promoter, Contains the α-actin promoter coupled to the albumin promoter, PGK, and CMV enhancer (Doll, R., et al., 1996, Gene Ther., 3 (5): 437-47).

  Other components of the vector construct include DNA molecules (eg, endogenous sequences useful in homologous recombination), tissue-specific promoters, parent cells, which are optionally designed for site-specific integration. DNA molecules that can provide superior selection benefits, DNA molecules useful as labels for identifying transformed cells, negative selection systems, cell-specific binding agents (for cell targeting), cell-specific Internalization factors, transcription factors that enhance expression from the vector, and factors that allow the production of the vector.

  In one embodiment, the vector can include targeting DNA for site-specific integration. The targeting DNA is a nucleotide sequence that is complementary (homologous) to the region of interest, for example, the ABC1 gene region. Through homologous recombination, the DNA sequence to be inserted into the genome can be directed to the ABC1 gene by attaching it to the targeted DNA. DNA sequences for insertion include, for example, regions of DNA that can interact with or control the expression of ABC1 polypeptides, eg, flanking sequences. Thus, expression of the desired ABC1 polypeptide is greater than that of the ABC1 gene, rather than by using targeted DNA coupled with a DNA regulatory segment that provides an endogenous gene sequence with a recognizable signal for transcription of the ABC1 polypeptide. It is accomplished by transfection of DNA encoding itself (Sauer, Curr. Opin. Biotechnol., 5: 521-27 (1994); Sauer, Methods Enzymol., 225: 890-900 (1993)).

  In still other embodiments, regulatory elements can be included for controlled expression of the ABC1 gene in target cells. Such elements are switched on in response to an appropriate effector. Thus, a therapeutic ABC1 polypeptide can be expressed if desired. One conventional control means is a small molecule for dimerizing a chimeric protein containing a small molecule-binding domain and a domain that can initiate a biological process, such as a DNA-binding protein or a transcriptional activation protein. Includes the use of molecular demelizers or rapalogs (see PCT publication numbers WO 96/41865, WO 97/31898 and WO 97/31899). Protein dimerization can be used to initiate transgene transcription. Another suitable control means or gene switch involves the use of mifepristone (RU486), a progesterone antagonist. Binding of the modified progesterone receptor ligand-binding domain to the progesterone antagonist then activates transcription by forming dimers of two transcription factors that pass through the nucleus and bind DNA to DNA. The ligand-binding domain is modified to eliminate the ability of the receptor to bind to the natural ligand. Modified steroid hormone receptor systems are further described in US Pat. No. 5,364,791 and PCT Publication Nos. WO96 / 40911 and WO97 / 10337. Yet another control means uses a positive tetracycline-controllable transactivator. This system results in a mutated tet repressor protein (DNA-binding domain (reverse tetracycline-regulated transactivator protein) linked to a polypeptide that activates transcription, i.e., it becomes tet operator in the presence of tetracycline. Such systems are described in US Patent Nos. 5,464,758, 5,650,298 and 5,654,168. Additional expression control systems and nucleic acid constructs are described in US Pat. Nos. 5,741,679 and 5,834,186.

A viral delivery vector containing an ABC1 polynucleotide can be made target specific by altering the viral coat so that it contains a ligand specific for another molecule found in the target cell. This allows the vector to bind to the desired cell type. The ligand can be any compound of interest that specifically binds to a molecule found in the target cell, such as a cell-surface receptor. Preferably, the receptor is found exclusively in the target cell and not in other cells. For example, a ligand for scavenger receptor A can be used to direct the viral delivery vector to macrophage cells. Alternatively, the viral coat can be altered so that it contains an antibody or antibody fragment such as Fab or F (ab ′) ₂ that recognizes and binds to an antigenic epitope on the target cell. The viral coat can be altered by inserting additional polynucleotide coating ligands into the viral genome. One skilled in the art knows other specific polynucleotide sequences that can be inserted into the viral genome to allow target-specific delivery of vectors containing ABC1 polynucleotides.

  In addition, as described above, a naked ABC1 polynucleotide can be administered. ABC1 polynucleotides and recombinant ABC1 expression vectors can be administered as pharmaceutical compositions. Such a composition comprises an effective amount of an ABC1 polynucleotide or recombinant ABC1 expression vector as defined herein above and a pharmaceutically acceptable formulation suitably selected for its mode of administration. Suitable formulation materials are preferably non-toxic to the recipient at the concentrations used and are described below.

  A pharmaceutical composition comprising an ABC1 polynucleotide or an ABC1 recombinant expression vector is, for example, the pH, osmolarity, viscosity, transparency, color, isotonicity, odor, sterility, stability, dissolution or release of the composition, Formulation materials can be included to modify, maintain, or maintain the rate of adsorption or penetration. Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine), antimicrobial agents, and antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite). Buffer (such as borate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids), bulking agent (such as mannitol or glycine), (ethylenediaminetetraacetic acid (EDTA)) Chelating agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin) complexing agents, fillers, monosaccharides, disaccharides and (glucose, mannitol or dextrin) Other carbohydrates, such as (serum albumin) Proteins (such as gelatin or immunoglobulin), colorants, flavors and diluents, emulsifiers, hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight polypeptides, (such as sodium) salt-forming counterions, (benzamide chloride) Preservatives (such as luconium, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide), solvents (such as glycerin, propylene glycol or polyethylene glycol), mannitol or sorbitol Sugar alcohol, suspending agent, (pluronic; PEG; sorbitan ester; polysorbate such as polysorbate 20 or polysorbate 80; trilone; tromethanine; reticin; A surfactant or wetting agent (such as terol or tyloxafal), a stability enhancer (such as sucrose or sorbitol), an isotonicity enhancer (such as an alkali metal halide (preferably sodium chloride or potassium- or mannitol, sorbitol), Delivery vehicles, diluents, excipients and / or pharmaceutical adjuvants, see Remingotn's Pharmaceutical Sciences (18th Ed., AR Gennaro, Ed., Mack Publishing Company 1990).

  Pharmaceutically active compounds (ie, ABC1 polynucleotides or ABC1 vectors) can be processed according to conventional pharmaceutical methods to produce pharmaceutical agents for administration to patients, including humans and other mammals. it can. Thus, pharmaceutical compositions comprising ABC1 polynucleotides or ABC1 recombinant expression vectors can be made in solid or liquid form (including granules, powders or suppositories) (eg, solutions, suspensions or emulsions). Solid dosage forms for oral administration can include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound can be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms can contain, in normal practice, additional materials other than inert diluents, for example, lubricants such as magnesium stearate. In the case of capsules, tablets and pills, the dosage forms can also contain buffering agents. Tablets and pills can additionally be prepared with an enteric coating.

  Liquid dosage forms for oral or parenteral administration contain pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs (including inert diluents commonly used in the art such as water). . Such compositions can also contain wetting agents, sweetening, flavoring, and perfuming agents. For example, a suitable carrier for injection may be water, saline or artificial cerebrospinal fluid, possibly supplemented with a composition for parenteral administration, usually other substances. Neutral buffered saline or saline mixed with serum albumin are further exemplary carriers. Other exemplary pharmaceutical compositions include Tris buffer at about pH 7.0-8.5, acetate buffer at about pH 4.0-5.5, which further includes sorbitol or a suitable alternative Can do.

  Administration methods for treating cardiovascular disease with compositions comprising ABC1 polynucleotides or ABC1 expression vectors include cardiovascular disease type, patient age, weight, gender and medical condition, severity of disease, route of administration and use Based on a variety of factors, including the particular compound to be. Thus, although the method of administration can vary widely, it can be routinely determined using standard methods. For example, the amount of ABC1 polynucleotide or ABC1 expression vector to be administered is an amount sufficient to increase cholesterol efflux from cells of a mammalian subject. Such amount can be determined, for example, by measuring a subject's plasma HDL-cholesterol levels before and after administration of ABC1 polynucleotide or ABC1 expression vector. A sufficient amount of ABC1 polynucleotide or ABC1 expression vector is an amount that increases plasma HDL-cholesterol levels in a subject. Therefore, the clinician can determine the dose and modify the administration route to obtain the optimal therapeutic effect. Typical dosages can range from about 0.1 mg / kg to about 100 mg / kg or more based on the factors described above.

  The frequency of administration will depend on the pharmacokinetic parameters of the ABC1 polynucleotide or vector in the formulation to be used. Typically, the clinician will administer the composition until a dosage is reached that achieves the desired effect. Accordingly, the composition may be administered as a single dose over time, in two or more doses (which may or may not contain the same amount of the desired molecule), or continuously via a discolored device or catheter. Can be administered as a general infusion. Further modifications of the appropriate dosage are routinely made by those skilled in the art and are within the scope of work routinely done by those skilled in the art. Appropriate dosages can be ascertained by using appropriate dose-response data.

  Mammalian subject cells can be transfected in vivo, ex vivo or in vitro. In vivo administration of ABC1 polynucleotides or recombinant vectors containing ABC1 polynucleotides to target cells can be accomplished using any of a variety of techniques known to those skilled in the art. . For example, US Pat. No. 5,672,344 describes an in vivo virus-mediated gene transfer system comprising a recombinant neurotrophic HSV-1 vector. Said compositions of ABC1 polynucleotides and recombinant ABC1 vectors can be transfected in vivo by oral, buccal, parenteral, rectal or topical administration, as well as by inhalation spraying. The term “parenteral” as used herein includes subcutaneous, intravenous, intramuscular, intrasternal, infusion technique or intraperitoneal.

For oral administration, pharmaceutical compositions containing ABC1 polynucleotides or recombinant ABC1 vectors can be in the form of capsules, tablets, suspensions or liquids, for example. The pharmaceutical composition is preferably made in the form of a dosage unit containing a given amount of DNA or viral vector particles. For example, they can contain an amount of about 10 ³ -10 ¹⁵ viral particles, preferably about 10 ⁶ -10 ¹² viral particles. Appropriate daily doses for humans or other mammals can vary widely depending on the condition of the patient and other factors, but can again be determined using routine methods.

  A pharmaceutical composition containing ABC1 DNA or a recombinant ABC1 vector can also be administered rectally. Suitable suppositories for rectal administration of vectors are solid at room temperature, but liquid at rectal temperature, and therefore suitable non-irritating agents such as cocoa butter and polyethylene glycol that melt in the rectum and release the vector. It can be prepared by mixing the excipient and the vector.

  The pharmaceutical composition can also be formulated for inhalation. For example, an ABC1 polynucleotide or vector can be formulated as a dry powder for inhalation. ABC1 polynucleotide or vector inhalation solutions can also be formulated with a propellant for aerosol delivery. In yet another embodiment, the solution can be atomized.

  A pharmaceutical composition containing ABC1 DNA or recombinant ABC1 vector can also be injected. Injection preparations such as sterile injectable aqueous or oleaginous suspensions can be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. . A particular suitable carrier for parenteral injection is sterile distilled water formulated ABC1 polynucleotide or vector as a suitably stored sterile isotonic solution. Among the acceptable carriers and solvents that can be used are Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conveniently employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can be used in the preparation of injectables. Yet another formulation can include the formulation of the desired ABC1 molecule in an agent such as an injectable microsphere, biodegradable particle, polymer compound, bead or liposome, which can be controlled or retained for the ABC1 product. (See, eg, PCT / US93 / 00829; Epstein et al., Proc. Natl. Acad. Sci., 82: 3688-3692 (1985)).

  In addition, a pharmaceutical composition containing ABC1 DNA or recombinant ABC1 vector can be administered locally. For topical administration, the vector may comprise 0.001% to 10% w / w of the formulation, for example 1% to 2% by weight, which is preferably as much as 10% w / w of the formulation, preferably It may contain 5% w / w or less, more preferably 0.1% to 1%. Formulations suitable for topical administration include liquid or semi-liquid formulations (eg, liniments, lotions, ointments, creams, or pastes) suitable for penetration through the skin and drops suitable for administration to the eyes, ears or nose. . Appropriate local doses of the active ingredients of the vectors of the invention are administered 1 to 4 times, preferably 2 or 3 times daily.

  While the nucleic acids and / or vectors of the invention can be administered as the only active pharmaceutical agent, it can also be used in combination with one or more vectors of the invention or other agents. When administered as a combination, the therapeutic agents can be formulated as separate compositions that are given the same number or different times, or the therapeutic agents can be given as a single composition.

  In another embodiment of the invention, the target cells are transfected in vivo by the discoloration of the “producer cell line” adjacent to the target cell population (Culver, K., et al., 1994, Hum. Gene Ther. , 5 (3): 343-79; Culver, K, et al., Cold Spring Harb. Symp. Quant. Biol., 59: 687-90; Oldfield, E., 1993, Hum Gene Ther., 4 (19: 39-69) The producer cell line produces a viral vector containing the ABC1 polynucleotide and its viral particles in close proximity to the target cells, ie, macrophage cells, preferably found in atherosclerotic lesions. It is made to emit. Some of the particles contact and infect the target macrophage cell, thus delivering ABC1 polynucleotide to the target macrophage cell, following the target cell infection, expression of ABC1 polynucleotide occurs, A functional ABC1 protein is provided.

  In another embodiment, the present invention provides a method of treating cardiovascular disease by X-vivo introduction of an ABC1 polynucleotide or a recombinant ABC1 expression vector. In such instances, cells, tissues or organs removed from the patient are exposed to the ABC1 composition, after which the cells, tissues or organs are subsequently returned to the patient and transplanted. For example, one method involves removing a blood sample from a subject with cardiovascular disease, making the sample rich in monocytes, and treating the isolated monocytes with an ABC1 polynucleotide. And optionally contacting with a target specific gene. If desired, monocyte cells can be treated with a growth factor such as GM-CSF to stimulate cell proliferation before reintroducing the cells into the subject. When reintroduced, the cell specifically targets the cell population from which it was originally isolated. Thus, the transport activity of ABC1 polypeptide can be used to promote cholesterol efflux in a subject.

  Another method of X-vivo administration involves introducing an ABC1 polynucleotide or a recombinant ABC1 vector into a mammalian subject by skin grafting of cells containing the virus. Preferably retroviruses are used in this method of administration. If a strong housekeeping gene promoter is used to drive transcription, cells of fibroblast origin can be used to achieve long-term expression of foreign genes in the implant. For example, a dihydrofolate reductase (DHFR) gene promoter can be used. Cells, such as fibroblasts, can be infected with virions containing genes that allow specific targeting, such as scavenger A, and retroviral constructs that contain ABC1 polynucleotides with a strong promoter. Infected cells can be embedded in a collagen matrix that can be transplanted into the connective tissue of the dermis in the recipient subject. As the retrovirus grows and escapes from the matrix, it will specifically infect the target cell population. In this way, transplantation results in an increased amount of cholesterol efflux activity in cells exhibiting transport disorders.

  In another embodiment, recombinant expression vectors comprising ABC1 polynucleotides can be administered using in vitro techniques, as described in US Pat. No. 5,399,346. For example, ABC1 polypeptides can be delivered to express ABC1 polypeptides by transplanting certain cells that have been genetically engineered using methods such as those described herein. In order to minimize the potential immune response in a patient to whom an ABC1 polypeptide is to be administered, such as can occur with administration of a foreign species polypeptide, the natural cells that produce the ABC1 polypeptide are of human origin and human It is preferred to produce ABC1 polypeptides. Thus, recombinant cells producing ABC1 polypeptide are preferably transformed with an expression vector containing a gene encoding a human ABC1 polypeptide. The cell can be autologous or heterogas. If desired, the cells can be immortalized. To reduce the chance of an immune response, cells can be encapsulated to avoid invasion of surrounding tissue. Encapsulating materials typically allow for rewarding protein products, but are biocompatible, semi-permeable to prevent cell destruction by the patient's immune system or other harmful factors from surrounding tissues A polymer container or membrane. Using the methods described above, the transfected cells can be administered to the patient.

  Techniques for live cell encapsulation are known in the art, and the preparation of encapsulated cells and their transplantation in patients can be accomplished routinely. For example, Baetge et al. (PCT Publication Nos. WO95 / 05452 and PCT / US94 / 09299) describe membrane capsules containing genetically engineered cells for effective delivery of biologically active molecules. The capsule is biocompatible and can be easily recovered. The capsule was transfected with a recombinant DNA molecule comprising a DNA sequence encoding a biologically active molecule operably linked to a promoter that is not subject to down regulation in vivo upon implantation into a mammalian host. Encapsulate the cells. The device allows delivery of molecules from living cells to specific sites within the recipient. In addition, see U.S. Pat. Nos. 4,892,538; 5,011,472; and 5,106,627. A system for encapsulating living cells is described in PCT International Publication No. WO 91/10425 (Aebischer et al.). See also PCT Publication No. WO 91/10470 (Aebischer et al.); Winn et al., 1991, Expert. Neurol. 113: 322-29; Aebischer et al., 1991, Exper. Neurol. 111: 269-75; and Tresco et al., 1992, ASAIO 38: 17-23.

Another delivery system for a polynucleotide encoding ABC1 is a “non-viral” delivery system. Or proposed techniques DNA- ligand complexes used in gene therapy, adenovirus - ligand -DNA complexes, direct injection of DNA, CaPO ₄ precipitation, gene gun techniques, electroporation, lipofection and colloidal dispersion (Mulligan, R., 1993, Science, 260 (5110): 926-32). Any of these methods are widely available to those skilled in the art and would be suitable for use in the present invention. Other suitable methods are available to those skilled in the art and it should be understood that the present invention can be achieved using any of the available methods of transfection. Several such methods have been utilized by those skilled in the art and have met with varying degrees of success (Mulligan, R., 1993, Science, 260 (5110): 926-32).

  Preferably, the non-viral delivery system is a colloidal dispersion system. Colloidal dispersions include macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles and liposomes. A preferred colloidal system of the present invention is a liposome, which is an artificial membrane vesicle useful as a delivery vehicle in vitro and in vivo. Liposomes are self-assembling colloidal particles in which a lipid bilayer composed of amphipathic molecules such as phosphatidylserine or phosphatidylcholine is separated from a surrounding medium such that the lipid bilayer surrounds the hydrophilic interior. Encapsulate the part. Single lamellar or multilamellar liposomes can be constructed so that the interior contains the desired chemical, drug, or isolated DNA molecule as in the present invention. For example, large single lamellar vesicles (LUVs) ranging in size from 0.2-4.0 μm encapsulate a substantial percentage of aqueous buffers containing large macromolecules such as RNA, DNA and intact virions. It has been shown that it can. Once encapsulated within an aqueous interior, these macromolecules can be delivered to mammalian cells in a biologically active form (Fraley, R. and Papahadjopoulos, D. 1981, Trends Biochem. Sci., 6: 77-80). In order for liposomes to be effective gene transfer vehicles, the following characteristics should exist: (1) Encapsulate genes of interest with high efficiency without compromising their biological activity. (2) preferentially and substantially bind to the target cell compared to the non-target cell; (3) the aqueous content of the vesicle is delivered to the cytoplasm of the target cell with high efficiency; and (4 ) The genetic information is expressed accurately and effectively (Manno, R. et al., 1988, Biotechniques, 6 (7): 682-90).

  The composition of the liposome is usually a combination of phospholipids, particularly high phase transition temperature phospholipids, usually combined with steroids, especially cholesterol. Other phospholipids or other lipids can also be used. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations. Examples of lipids useful in the production of liposomes include phosphatidyl compounds such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides and gangliosides. Particularly preferred is diacylphosphatidylglycerol, where the lipid moiety contains 14-18 carbon atoms, especially 16-18 carbon atoms, and is saturated. Exemplary phospholipids include egg phosphatidylcholine, dipalmitryl phosphatidylcholine and distearoyl phosphatidylcholine.

Liposome targeting has been classified based on anatomical and mechanical factors. Anatomical classification is based on the level of selectivity, eg, organ-specificity, cell-specificity and organelle-specificity. Mechanical targeting can be distinguished based on whether it is passive or active. Passive targeting takes advantage of the natural tendency of liposomes distributed in cells of the reticuloendothelial cell line (RES) in organs containing sinusoidal capillaries. On the other hand, active targeting is a naturally occurring site of localization by coupling liposomes to specific ligands such as polyclonal or monoclonal antibodies, sugars, glycolipids or proteins, or by changing the composition or size of the liposomes. Modifying the liposomes by achieving targeting to other organs and cell types. Preferably, the ligand is a polyclonal or monoclonal antibody that can be used to target liposomes to specific cell-surface ligands. The ligand can also be an antibody fragment such as Fab or F (ab ′) ₂ so long as it effectively binds to an antigenic epitope on the target cell. Preferably, the antibody or antibody fragment recognizes an antigen found exclusively in the target cell. For example, certain antigens specifically expressed on macrophage cells, such as scavenger receptor A, can be developed for the purpose of targeting antibody-ABC1 liposomes directly to macrophage cells.

  Numerous techniques can be used to covalently attach either polyclonal or monoclonal antibodies to the liposome bilayer. Lipid groups can also be incorporated into the lipid bilayer of the liposome to maintain the targeting ligand stably associated with the liposome bilayer. In addition, various linking groups can be used to conjugate the lipid chain to the targeting ligand.

  The studies presented here have shown that ligands for nuclear receptors can increase cholesterol efflux. Accordingly, in another embodiment, the present invention comprises administering to a mammalian subject at least one ligand for a nuclear receptor in an amount sufficient to increase cholesterol efflux from cells in the mammalian subject. A method suitable for increasing cholesterol efflux from the cells is provided. Using the method of formulating and administering a pharmaceutical composition, a pharmaceutical composition comprising a ligand for a nuclear receptor can be prepared and administered. A sufficient amount of nuclear receptor ligand is that amount of ligand that increases cholesterol efflux. Such amount can be determined by measuring cholesterol efflux before and after administration of various doses of ligand and determining the dose that results in increased cholesterol efflux. Cholesterol efflux can be measured using the assay described above.

  Nuclear receptors are ligand-activated transcription factors that play a critical role in vertebrate development and adult physiology by introducing the effects of small lipophilic hormones into the transcriptional response. Includes peroxisome proliferator-activated receptor (PPAR), liver X receptor (LXR), retinoid X receptor (RXR), farnesoid X receptor (FXR), and steroid and xenobiotic receptor (SXR) There are several families of nuclear receptors. The PPAR family includes three closely related gene products PPARα, PPARγ and PPARβ / δ. PPARσ has been shown as a key regulator of intracellular and extracellular lipid metabolism. When bound to fatty acids, PPARα stimulates peroxisome proliferation and induces the synthesis of several enzymes involved in fatty acid β-oxidation. The PPARα receptor is also a molecular target for fibrates, drugs prescribed for lowering triglyceride levels (Isseman et al., Nature, 347,645 (1990)). Fibrates act as PPAR ligands that regulate the transcription of a large number of genes that affect lipoprotein and fatty acid metabolism. In addition, PPARγ ligands such as those belonging to the class of thiazolidinedione compounds have been shown to increase HDL and reduce triglyceride levels in humans.

  LXR is an oxysterol receptor that regulates catabolism of excess cholesterol. LXRα has been shown to bind as a heterodimer with RXR to a DNA response element in the CYP7a gene that encodes an enzyme responsible for the rate-limiting step in the conversion of cholesterol to bile acids. Research has shown that ingestion of cholesterol-rich diets causes mice lacking LXRα to accumulate enormous amounts of cholesterol esters in their livers because they cannot increase transcription of the CYP7a gene in response to diet cholesterol. Show iru (Peet et al., Cell, 93: 693 (1998)). Further studies in LXRα null mice indicate that LXRα is also involved in the regulation of several other genes that participate in cholesterol and fatty acid homeostasis. The biological role of the closely related nuclear receptor LXRβ expressed in several tissues and activated by the same oxysterol as LXRα remains unclear (Song et al., Proc. Natl. Acad. Sci., 91 : 10809 (1994); Seol, Mol. Endocrnol., 9:72 (1995)).

  FXR, which is revolutionarily related to LXRα, is also involved in cholesterol homeostasis. Like LXRα, FXR functions as a heterodimer with the RXR receptor (Schwartz et al., Curr. Opin. Lipidol., 9: 113 (1998); Vlahsetic et al., Gastroenterology, 113: 949 (1997)). FXR is activated by synthetic retinoid TTNPB and superphysiological concentrations of all trans retinoic acid (Xavacki et al., Proc. Natl. Acad. Sci., 94: 70-0 (1997)). Recent studies indicate that FXR is a nuclear bile acid receptor. First, FXR is abundantly expressed in tissues through which bile acids circulate, including the liver, intestine and kidney (Seol et al., Mol. Endocrinol., 9:72 (1995); Forman et al., Cell, 81: 687 (1995)). FXR has also recently been shown to act as a receptor for several bile salts at physiological concentrations, of which chenodeoxycholic acid (CDCA) is the best (Kliewer et al., Science, 284: 757-284 ( 1999); Makishima et al., Science, 284: 1362-1363 (1999)). CDCA is known to regulate the expression of several genes that participate in bile salt homeostasis, including those encoding CYP7a and intestinal bile acid-binding proteins.

  As described in detail in Example 13, ligands for LXR, RXR and PPAR nuclear receptors have been shown to increase apoAI-induced cholesterol efflux in cholesterol-loaded mouse macrophage cells. For example, administration of 9 cis-retinoic acid (30 ng / ml) resulted in an approximately 3-fold increase in apo AI-induced cholesterol efflux in these cells. Similarly, administration of 22 (R) hydroxycholesterol (5 μ / ml) resulted in an almost 3-fold increase in apoAI-induced cholesterol efflux. Cells ingested fenfibrate (3 μg / ml) produced an almost 2-fold increase in cholesterol efflux. These results indicate that the nuclear receptor is modulated to increase the rate of apolipoprotein-mediated cholesterol efflux from macrophages. Furthermore, as described in Example 13, 9-cis-RA mediated cholesterol efflux from macrophage cells in a dose-dependent manner. Other nuclear receptor activators such as benzafibrate have been shown to increase cholesterol efflux (data not shown).

  Thus, in a method of increasing cholesterol efflux by administering a nuclear receptor ligand, the ligand is preferably selected from the group consisting of LXR, RXR, PPAR, FXR and SXR nuclear receptor ligands. In a preferred embodiment where LXR ligands are used to increase cholesterol efflux, the ligand is more preferably 20 (S) hydroxycholesterol, 22 (R) hydroxycholesterol, 24-hydroxycholesterol, 25-hydroxycholesterol, and 24 (S ), 25 epoxycholesterol LXR ligands. Most preferably, the LXR ligand is 20 (S) hydroxycholesterol. In a preferred embodiment of increasing cholesterol efflux using RXR ligands, the ligand is more preferably 9-cis retinoic acid, retinol, retinal, all-trans retinoic acid, 13-cis retinoic acid, acitretin, fenretinide, etretinate, It is selected from the group consisting of CD495, CD564, TTNN, TTNNPB, TTAB, and LDG1069. Most preferably, the RXR ligand is 9-cis retinoic acid. In another preferred embodiment using PPAR ligands to increase cholesterol efflux, the ligand is preferably selected from the class of thiazolidinedione compounds.

  In another preferred embodiment, more than one nuclear receptor ligand is administered to the mammalian subject to increase cholesterol efflux. Preferably, when more than one nuclear receptor ligand is administered to a subject, the ligands are LXR and RXR ligands. More preferably, the nuclear receptor ligand is 20 (S) hydroxycholesterol and 9-cis retinoic acid.

  In yet another embodiment, the present invention comprises the step of administering eicosanoids to a mammalian subject in an amount sufficient to increase cholesterol efflux, in a mammalian subject, Provide a suitable way to increase. A sufficient amount of eicosanoid that can be prepared and administered using the above method of formulating and administering a pharmaceutical composition and comprising an eicosanoid is an amount that increases cholesterol efflux. Such amounts can be determined by measuring cholesterol efflux before and after administration of various doses of eicosanoids and measuring the dose that results in increased cholesterol efflux. Cholesterol efflux can be measured using the assays and methods described above.

  As described in Example 14, eicosanoids have been shown to increase apo AI-induced cholesterol efflux in cholesterol-loaded mouse macrophage cells. For example, administration of PGI2 (25 nm) caused an almost 2-fold increase in apo-induced cholesterol efflux in these cells. Similarly, administration of PGE1 (nM) resulted in an approximately 3-fold increase in apoAI-induced cholesterol efflux. These results indicate that eicosanoids can increase the rate of apolipoprotein-mediated cholesterol efflux from macrophages. Thus, in a preferred embodiment, the eicosanoid is selected from the group consisting of prostaglandin E2, prostacyclin (prostaglandin I2) and prostaglandin J2 eicosanoid.

(Methods and compounds for increasing ABC1 expression / activity))
Given that ABC1 functions to promote cholesterol efflux, one way to increase cholesterol efflux is to increase cellular expression of ABC1. Accordingly, the present invention provides a method suitable for increasing cholesterol efflux from a cell by administering to the mammalian subject a therapeutic amount of a compound that increases ABC1 expression in the cell in the mammalian subject. A therapeutic amount of a compound is that amount of a compound that increases ABC1 expression. Such amounts can be determined by measuring ABC1 gene expression before and after administration of various doses of the compound and measuring the dose that results in increased ABC1 gene expression. ABC1 gene expression is measured by obtaining a blood sample from a subject, isolating a monocyte population, and measuring the concentration of ABC1 mRNA using methods known in the art and described herein, such as RT-PCR. be able to.

  In one preferred embodiment, the method comprises administering a cAMP analog to increase ABC1 gene expression. As shown in FIG. 8, cAMP increases ABC1 mRNA expression almost 10-fold in normal fibroblasts. Preferably, the cAMP analog is selected from the group consisting of 8-bromo cAMP, N6-benzoyl cAMP and 8-thiomethyl cAMP. In another preferred embodiment, the method comprises increasing cAMP synthesis to increase ABC1 gene expression. Preferably the compound is forskolin. In yet another preferred embodiment, the method comprises administering a compound that inhibits cAMP degradation and increases ABC1 gene expression. Examples of such compounds are phosphodiesterase inhibitors. Preferably, the phosphodiesterase inhibitor is rolipram, theophylline, 3-isobutyl-1-methylxanthine, R020-1724, vinpocetine, zaprinast, dipyridamole, milrinone, amrinone, pimobendan, cilostamide, enoximone, peroximone, and vesnarinone phosphodiesterase inhibitor Selected from the group consisting of:

  In another preferred embodiment, the method comprises administering to the mammalian subject a ligand for a nuclear receptor in an amount sufficient to increase ABC1 gene expression. As described in Example 17 and shown in FIG. 12, ligands for nuclear receptors can upregulate ABC1 gene expression. Transfection experiments with pAPR1 containing the luciferase reporter gene under the control of the ABC1 promoter showed that the ABC1 promoter is activated in the presence of ligands for LXR and RXR nuclear receptors. Specifically, macrophage cells transfected with pAPR1 increased 19-fold increase in luciferase reporter activity in the presence of 20OH-chol, 16-fold increase in luciferase activity in the presence of 9-cis RA, and compared to EtOH control This resulted in a 280-fold increase in luciferase activity in the presence of both ligands. The results indicate that both sterols and retinoids induce a strong transcriptional response from the ABC1 promoter. Furthermore, there is a clear synergy between the two classes of compounds, as can be seen by the dramatic increase in luciferase activity found in cells treated with both ligands. According to the method of the present invention, preferably the ligand is selected from the group consisting of LXR, RXR, PPAR, FXR and SXR ligands.

  In addition to increasing the cellular level of ABC1 protein, reverse cholesterol transport can be facilitated by enhancing the activity of ABC1 protein. Thus, in another embodiment, the invention includes administering to a mammalian subject a therapeutic amount of a compound that increases ABC1 activity in an amount sufficient to increase cholesterol efflux. Methods are provided that are suitable for increasing cholesterol efflux from cells in animal subjects. Pharmaceutical compositions comprising such compounds can be prepared and administered using the methods described above for formulating and administering pharmaceutical compositions. A therapeutic amount of a compound is that amount of the compound that increases cholesterol efflux. Such an amount can be determined by measuring cholesterol efflux before and after administration of various doses of the compound and measuring the dose that results in an increase in cholesterol efflux using the methods described above. To determine whether the increase in cholesterol efflux is due to an increase in ABC1 activity, the amount of ABC1 protein present in the cell sample before and after administration of the compound is measured using the methods described herein (Example 11). reference). For both measurements (ie before and after administration of the compound), the amount of cholesterol efflux activity is divided by the concentration of ABC1 protein to determine the amount of cholesterol activity found at the standard concentration of ABC1 protein. The observed increase in cholesterol activity normalized to protein concentration indicates that the increase is due to an increase in ABC1 activity.

(Methods for identifying therapeutic compounds)
Another aspect of the invention relates to a method of screening a compound to determine whether the compound modulates ABC1 gene expression (ie, upregulation or downregulation). Such compounds would be useful in the development of therapeutic compounds that increase ABC1 expression, thereby promoting cholesterol efflux and increasing blood levels of HDL-cholesterol. Accordingly, methods are provided for identifying compounds that may be useful in the treatment of cardiovascular disease. In one embodiment, the present invention provides (a) operably linking an expression modulating portion of a mammalian ABC1 gene and a reporter cDNA to produce a recombinant reporter construct; (b) a recombinant reporter construct; Transfecting a population of host cells; (c) assaying the level of reporter gene expression in a sample of the transfected host cells; (d) contacting the transfected cells with a test compound to be screened; e) assaying the level of reporter gene expression in a sample of host cells transfected after contact with the test compound; then (f) relative changes in the level of reporter gene expression caused by exposure to the test compound And thereby determining ABC1 expression modulating activity. It provides a method of screening a test compound per ABC1 expression modulating activity, characterized in that.

  First, a recombinant reporter construct is constructed that includes a heterologous reporter gene operably linked to the expression modulating portion of the ABC1 gene. Davis et al., Basic Methods in Molecular Biology (1986); Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, (Cold Spring Harbor Press, Cold Sr. ABC1 expression-modulating polynucleotides and reporters using well-known ligation and cloning techniques, such as those in standard laboratory manuals described herein, including Current Protocols in Molecular Biology, (Wiley and SOns (1994)). The gene can be inserted into a vector. Alternatively, ABC1 expression modulating polynucleotides can be inserted into commercially available reporter constructs such as those described above. Any vector suitable for insertion of ABC1 polynucleotides and reporter genes can be used. The selected vector should function in the particular host cell used. Preferably, the vector is compatible with a mammalian host cell.

  Preferably, the expression-modulating part of the ABC1 gene is ABC1's 5 'flanking region containing ABC1 promoter activity. In one preferred embodiment, the expression modulating portion of the ABC1 gene comprises SEQ ID NO: 3. In another preferred embodiment, the expression modulating portion of the ABC1 gene comprises nucleotides 1-1532, 1080-1643, 1181-1634, 1292-1643, 1394-1643 or 1394-1532 of SEQ ID NO: 3. Also preferably, the heterologous reporter is selected from the group consisting of a luciferase, β-galactosidase, chloramphenicol acetyltransferase and a polynucleotide encoding a green fluorescent protein. More preferably, the heterologous reporter is a polynucleotide encoding a luciferase protein. In a particularly preferred embodiment, the recombinant reporter construct is pAPR1 as shown in FIG.

  The recombinant reporter construct is then transfected into a population of host cells. Recombinant reporter constructs can be introduced into host cells using any of the transfection methods described above and the methods described in Examples 8 and 15. For example, the reporter construct can be transduced using calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, any of the other known methods described. (See, for example, (Davis et al., Basic Methods in Molecular Biology (1986).) A host cell, when cultured under appropriate conditions, can synthesize any reporter polypeptide that can subsequently be measured. Preferably, the host cell is a mammalian host cell, more preferably the mammalian host cell is a macrophage, fibroblast, hepatocyte or intestinal cell, more preferably a host cell. Selected from the group consisting of RAW264.7 cells, Thp-1 cells and HepG2 cells, the concentration of reporter construct and the duration of transfection being varied depending on the transfection method and the type and concentration of the host cell used. Determination of the appropriate concentration of reporter construct and transfection time is within the skill of the artisan.

  Following transfection, a sample of transfected host cells not exposed to the test compound is assayed to determine the level of reporter gene expression. The level of reporter gene expression found in a sample of unexposed transfected host cells provides a control measure. Transfected host cells are lysed and the level of reporter gene expression is assayed using any of the methods well known in the art. The assay used to measure the level of reporter gene expression varies depending on the reporter construct used in the transfection. For example, if a luciferase reporter construct is used, the luciferase activity of the cell lysate is measured as light units using a luminometer as described in Example 15.

  Different samples of transfected host cells are contacted with the test compound to be screened and the level of reporter gene expression found in these cells is subsequently measured. Preferably, the transfected host cell is contacted with the test compound for about 4-48 hours. More preferably, the transfected host cell is contacted with the test compound for about 8-36 hours. Even more preferably, the transfected host cell is contacted with the test compound for about 24 hours. The same assay used to measure the level of reporter gene expression in unexposed (control) cells should be used to measure the level of reporter gene expression in cells exposed to the test compound.

  Finally, comparing the level of reporter gene expression found in unexposed control cells to the level of reporter gene expression found in cells exposed to the test compound, the test compound has ABC1 expression modulating activity Determine whether or not. If the level of gene expression in both cell samples is the same or nearly the same, the test compound does not modulate ABC1 gene expression. A higher level of reporter gene expression in cells exposed to the test compound relative to the level of reporter gene expression found in the control cells indicates that the test compound upregulates ABC1 gene expression. A lower level of reporter gene expression in cells exposed to the test compound relative to the level of reporter gene expression found in the control cells indicates that the test compound downregulates ABC1 gene expression.

  Another aspect of the invention relates to a method of screening a test compound to determine whether the compound promotes ABC1-mediated cholesterol efflux. Such a method comprises (a) assaying the level of cholesterol efflux in a sample of mammalian cells maintained during culture to determine a control level of cholesterol efflux, and (b) a test to screen mammalian cells. (C) assaying the level of cholesterol efflux in the cell sample after contact with the test compound; then (d) ABC1-dependent cholesterol efflux in the cell sample after contact with the test compound; Assaying the level, thereby determining whether the test compound promotes ABC1-mediated cholesterol efflux from cells in culture.

  The level of cholesterol efflux in a sample of cultured cells can be measured using methods known in the art and described herein (see Example 1). Cholesterol efflux can be measured using any mammalian cell that can be maintained in culture. The cells can be derived from primary cultures or from immortalized cell lines. For convenience, as described in Example 1, cells are immortalized by transfection with an amphotropic retrovirus containing a human papillomavirus 16 insert, oncogenes E6 and E7, and a vector with a selectable marker gene. be able to. Preferably, the cultured cells are fibroblasts, macrophages, hepatocytes or enterocytes. More preferably, the cultured cells are RAW264.7 cells.

  The level of cholesterol efflux in a sample of cells that have not been contacted with test cells is measured to provide a control level of cholesterol efflux. In addition, the level of cholesterol efflux in the sample of mammalian cells contacted with the test compound is measured to determine the amount of cholesterol efflux affected by the test compound. Alternatively, the level of ABC1-mediated cholesterol efflux affected by the test compound is determined by measuring the level of ABC1-mediated cholesterol efflux in a sample of mammalian cells contacted with the test compound. Preferably, the cells are contacted with the test compound about 8-24 hours before assaying for cholesterol efflux or ABC1-mediated cholesterol efflux. The level of ABC1-mediated cholesterol efflux can be assayed with an anti-ABC1 antibody that inhibits the activity of ABC1 upon binding. Alternatively, antisense ABC1 polynucleotides that inhibit ABC1 expression can be used to assay the level of ABC1-mediated cholesterol efflux. For example, an antisense ABC1 polynucleotide comprising SEQ ID NO: 57 can be used to assay the level of ABC1-mediated cholesterol efflux (see Example 7). Cells should be contacted with the anti-ABC1 antibody or antisense ABC1 polynucleotide simultaneously with the contact with the test compound and for the same duration.

  If the level of control cholesterol efflux is the same or nearly the same as the level of cholesterol efflux found in cells contacted with the test compound, the compound does not promote cholesterol efflux. An increase in the level of cholesterol efflux found in cells contacted with a test compound that is greater than the control level of cholesterol efflux indicates the amount of cholesterol efflux promoted by the test compound. The difference between cholesterol efflux found in cells contacted with test compound alone and cholesterol efflux found in cells contacted with test compound and anti-ABC1 antibody or antisense ABC1 polynucleotide was mediated through ABC1 Shows the amount of cholesterol efflux. For example, if the control level of cholesterol efflux is 1.0 and the level of cholesterol efflux found in cells in contact with the test compound is 1.1, the test compound promotes cholesterol efflux by 10%. . If the cholesterol efflux found in cells contacted with the test compound and anti-ABC1 antibody or antisense ABC1 polynucleotide is 1.0, the increase in cholesterol efflux caused by the test compound is totally ABC1-mediated. .

(Method for detecting susceptibility to coronary heart disease)
The present invention also relates to a method of detecting a comparative level of ABC1 gene or protein expression in a mammalian subject, including a human subject. Assuming the role of ABC1 in cholesterol efflux, morbidity against coronary heart disease in a subject using measurements of increased levels of ABC1 gene or protein expression in a mammalian subject relative to a predetermined standard level of ABC1 gene or protein expression Can show gender. Accordingly, the present invention provides: (a) obtaining a test cell sample from a mammalian subject; (b) assaying the level of ABC1 mRNA expression in the test cell sample; and (c) the level of ABC1 mRNA expression in the test cell sample; Comparing with a predetermined standard level of ABC1 mRNA expression, thereby detecting a comparative level of ABC1 gene expression in a mammalian subject, comprising detecting a comparative level of ABC1 gene expression in the mammalian subject Provide a way to do it.

  First, a test cell sample is obtained from a mammalian subject including a human subject. The test cell sample can be a blood sample enriched in a monocyte population. Monocytes can be enriched using well-known cell separation techniques based on, for example, cell size, cell density, or cell renewal. Next, the level of ABC1 mRNA expression in the test cell sample is assayed. The level of ABC1 mRNA expression can be assayed using any of the well-known methods for mRNA preparation and detection, including the methods described herein in Examples 2 and 9. For example, the concentration of ABC1 mRNA can be measured by reverse transcription polymerase chain reaction, Northern blot analysis or RNAse protection assay. ABC1 mRNA concentration should be normalized to the concentration of total mRNA found in the test cell sample. Finally, ABC1 expression in the test cell sample is compared to a predetermined standard level of ABC1 mRNA expression. A predetermined standard level of ABC1 mRNA expression can be obtained by measuring the average concentration and distribution of ABC1 mRNA found in a cell sample taken from a representative population of mammalian subjects, wherein The animal subject is the same species from which the test cell sample was obtained, and wherein the mammalian subject does not have coronary heart disease, Tangier disease, or other diseases associated with low HDL-cholesterol It is believed to have cholesterol efflux activity within the normal range (indicated by HDL-cholesterol levels within the normal range). Measurement of reduced levels of ABC1 mRNA expression in mammalian subject test cell samples relative to a pre-determined standard level of ABC1 mRNA expression can be used to indicate susceptibility to coronary heart disease in a mammalian subject.

  Similarly, detection of reduced levels of ABC1 protein can be used to indicate reduced ability to cholesterol efflux and susceptibility to coronary heart disease. Accordingly, another embodiment of the present invention provides a method for detecting a comparative level of ABC1 protein in a mammalian subject. Such methods include: (a) obtaining a test cell sample from a mammalian subject; (b) assaying the amount of ABC1 protein in the test cell sample; and (c) determining the amount of ABC1 protein in the test cell sample from the ABC1 protein. Comparing to a predetermined standard amount, thereby detecting a comparative level of ABC1 protein in the mammalian subject.

  The amount of ABC1 protein can be assayed using any of the well-known methods of measuring protein. Preferably, the amount of ABC1 protein is measured using an immunoassay. In one embodiment, the amount of ABC1 protein is measured by (a) contacting a cell sample with a population of anti-ABC1 antibodies and then (b) detecting the anti-ABC1 antibody associated with the cell sample. . For example, ABC1 protein can be contacted with antisera raised against a synthetic peptide corresponding to KNQTVVDAVLTSFLQDEKVKES located at the C-terminus as described in Example 11. Anti-ABC1 antibodies can be detected using a number of methods known in the art including, for example, Western blotting, immunoprecipitation and FACS, where the detection is radioactivity, colorimetric or fluorescent. This can be achieved using a label. One preferred method for measuring the amount of ABC1 protein in a cell sample is immunoprecipitation, wherein the biotinylated ABC1 protein is contacted with an anti-ABC1 antibody and the bound anti-ABC1 antibody is streptavidin horseradish peroxidase. To detect.

  The amount of ABC1 protein in the test cell sample is compared to a predetermined standard amount of ABC1 protein. A predetermined standard amount of ABC1 protein can be obtained by measuring the average concentration of ABC1 protein found in a cell sample taken from a population of mammalian subjects, wherein the mammalian subject is then tested. The cell sample is the same species from which the sample was obtained, and here the mammalian subject does not have coronary heart disease, Tangier disease or other diseases associated with low HDL-cholesterol (within the normal range) It is believed to have cholesterol efflux activity within the normal range (indicated by HDL-cholesterol levels).

(ABC1 antibody)
As used herein, “antibodies” (Abs) or “monoclonal antibodies” (Mabs) are capable of specifically binding to intact molecules as well as proteins (eg, Fab and F (ab ′) 2 fragments). And so on) including an antibody fragment. Fab and F (ab ′) 2 fragments lack the Fc fragment of intact antibody and can be removed more rapidly from the circulation and have lower non-specific tissue binding than intact antibody (Wahl et al., J. Nucl. Med). , 24, 316-325 (1983)). Thus, these fragments, as well as products of FAB or other immunoglobulin expression libraries, are preferred. Furthermore, the antibodies of the invention include chimeric, single chain and humanized antibodies.

  Further embodiments include humanized versions of chimeric antibodies, eg, murine monoclonal antibodies. Such humanized antibodies can be prepared by known techniques and provide the benefit of reduced immunogenicity when administered to humans. In one embodiment, the humanized monoclonal antibody comprises a murine antibody variable region (or just its antigen binding site) and a constant region derived from a human antibody. Alternatively, the humanized antibody fragment can comprise an antigen binding site of a murine monoclonal antibody and a variable region fragment derived from a human antibody (lack of an antigen binding site). Techniques for the production of chimeras and more elaborate monoclonal antibodies are described by Riechmann et al., (Nature 332: 323, 1988), Liu et al. (PNAS 84: 3439, 1987), Larick et al., (Bio / Technology 7: 934, 1989). And those described by Winter and Harris (TIPS 14: 139, May 1993).

  One method of producing an antibody comprises a non-human animal, such as a transgenic mouse, comprising a polynucleotide comprising SEQ ID NO: 1, a polynucleotide comprising nucleotides 291-7074 of SEQ ID NO: 1, or SEQ ID NO: 1. Immunizing with a polypeptide translated from a polynucleotide comprising a nucleotide sequence having at least 90% identity to the polynucleotide, whereby an antibody directed against the polypeptide translated from the described polynucleotide Including being created in animals. Techniques have been developed to create human antibodies in non-human animals. The antibody can be partially human or preferably fully human. Non-human animals (such as transgenic mice) into which genetic material encoding one or more immunoglobulin chains has been introduced can be used. Such transgenic mice can be genetically modified in various ways. As a result of gene scanning, it is possible to obtain human immunoglobulin polypeptide chains that have replaced endogenous immunoglobulin chains in at least some (preferably substantially all) antibodies produced by animals upon immunization. Provided herein are antibodies produced by immunizing a transgenic animal with a polypeptide translated from any of the described polynucleotides.

  Mice have been prepared in which one or more endogenous immunoglobulin genes have been inactivated by various means. Human immunoglobulin genes have been introduced into mice to replace inactivated mouse genes. Antibodies produced in animals integrate human immunoglobulin polypeptide chains encoded by human genetic material introduced into the animal. Examples of techniques for production and use of such transgenic animals are provided in US Pat. Nos. 5,814,318, 5,569,825 and 5,545,806, which are incorporated herein by reference. Are listed.

  Monoclonal antibodies can be produced by conventional techniques, for example, by immortalizing spleen cells harvested from the transgenic animal after completion of the immunization schedule. Hybridomas can be produced by fusing spleen cells with myeloma cells by conventional techniques.

  A method of producing a hybridoma cell line immunizes such a transgenic animal with an immunogen comprising at least 7 consecutive amino acid residues of a polypeptide translated from one of the described polynucleotides; Harvesting the spleen cells from the animal; fusing the harvested spleen cells to a myeloma cell line, thereby creating a hybridoma cell; then monoclonals that bind to the translated polypeptide from one of the described polynucleotides Identifying the hybridoma cell line producing the antibody. Such hybridoma cell lines and monoclonal antibodies produced therefrom are included in the present invention. Monoclonal antibodies secreted by the hybridoma cell line are purified by conventional techniques.

  The antibody can inhibit the activity of ABC1 polypeptide upon specific binding to ABC1 polypeptide. Preferably, the antibody inhibits the cholesterol transport activity of ABC1 polypeptide upon binding. Such antibodies can be made by immunizing non-human animals with a polypeptide corresponding to a region essential for cholesterol transport. The antibody can be tested to determine whether it inhibits cholesterol efflux using any of the described cholesterol efflux assays. Such inactivated antibodies can be used in in vitro assays such as any of the cholesterol efflux assays described herein to determine whether a test compound promotes ABC1-mediated cholesterol efflux. it can. Inactivated antibodies can also be used in in vitro assays to detect comparative levels of ABC1 protein in mammalian subject cells. The inactivated antibody is useful in kits suitable for screening compounds to determine whether the compound modulates ABC1-dependent cholesterol efflux.

(Kit for identification of therapeutic compounds)
The present invention also includes a kit suitable for screening a compound in order to measure the ABC1 expression modulation activity of the compound. The kit comprises a recombinant reporter construct comprising a reporter cDNA operably linked to an expression modulating portion of a mammalian ABC1 gene as a separately packaged reagent in an amount sufficient to perform at least one assay. Including. Directives for the use of packaged reagents are also typically included. The expression modulating portion of the mammalian ABC1 gene contains a 5 'flanking sequence. In one preferred embodiment, the expression modulating portion of the mammalian ABC1 gene comprises SEQ ID NO: 3. In one preferred embodiment, the expression modulating portion of the mammalian ABC1 gene comprises nucleotides 1-1532, 1080-1643, 1181-1634, 1292-1643, 1394-1643 or 1384-1532 of SEQ ID NO: 3. The reporter cDNA can be any suitable reporter gene. In a particularly preferred embodiment, the recombinant reporter construct is pAPR1. In another embodiment, the kit further comprises a means for detecting the reporter protein. Thus, the kit includes reagents such as buffers and substrates used in reporter protein detection.

  As used herein, the term “package” refers to glass, plastic, (eg, polyethylene, polypropylene or polycarbonate), paper, which can hold the recombinant vector of the present invention within fixed limits. , Solid matrix or material such as foil. Thus, for example, the package can be a glass vial used to contain milligram quantities of a contemplated vector.

  The “instruction for use” typically includes at least the reagent concentration or relative amount of reagent and sample to be mixed, maintenance time for reagent / sample mixing, temperature, buffer conditions, etc. Contains a touchable expression describing one assay method parameter.

  In addition, the present invention includes kits suitable for screening compounds to determine whether the compounds modulate ABC1-dependent cholesterol efflux. In one embodiment, the kit comprises inactivated anti-ABC1 antibody as a separately packaged reagent in an amount sufficient to perform at least one assay. Directives for the use of packaged reagents are also typically included.

  In another embodiment, the kit comprises an amount of antisense ABC1 oligonucleotide sufficient for at least one assay and instructions for use. Preferably, the antisense ABC1 oligonucleotide comprises SEQ ID NO: 53.

(Microarray)
It will be appreciated that DNA microarray technology can be utilized in accordance with the present invention. A DNA microarray is a miniature high-density array of nucleic acids located on a solid support, such as glass. Each cell or element in the array contains multiple copies of a single nucleic acid species that serves as a target for hybridization with a complementary nucleic acid sequence (eg, mRNA). In expression profiling using DNA microarray technology, mRNA is first extracted from a cell or tissue sample and then enzymatically converted to fluorescently labeled cDNA. This material is hybridized to the microarray and unbound cDNA is removed by washing. The expression of the distinct genes represented on the array is then visualized by quantifying the amount of labeled cDNA specifically bound to each labeled nucleic acid molecule. In this way, the expression of thousands of genes can be quantified in a high-throughput parallel method from a single sample of biological material.

  This high-throughput expression profiling includes, but is not limited to: identification and validation of ABC1 disease-related genes as targets with therapeutic agents; molecular toxicology of related ABC1 molecules and their inhibitors; for clinical trials A broad range of ABC1 molecules of the present invention, including stratification of populations of surrogate markers and their creation; and enhancement of related ABC1 polypeptide small molecule drug discovery by helping to identify selective compounds in high-throughput screening Have application.

  As discussed herein in Example 2, a method has been developed that uses samples of RNA from cells of individuals with genetic abnormalities and compares them with RNA from normal individuals. Historically, identification of the cause of inherited disease is to identify suspected genes within millions of base pair intervals that have been shown to be closely linked to defects in genetic experiments (linkage analysis) From several years of biochemical analysis, or more recently, several years of genetic mapping and positional cloning. Most notably, the use of multigene expression analysis via a “gene chip” can reform the pace of such discovery. Comparing the expression of RNA samples from cells from an abnormal individual with a genetic disease vs. RNA samples from normal individuals is that the corresponding mRNA is lost in the abnormal disease cells and is severely underexpressed or Genes that are severely overexpressed can be quickly revealed.

  As used herein, the term “individual” refers to a living organism having RNA, such as a mammal or a plant. This method is preferably used to identify the source of human genetic abnormalities. More preferably, the method is useful for detecting genetic sources of human heart and cardiovascular disorders, such as identifying ABC1 as a genetic defect in Tangier disease.

  As used herein, the term “abnormality” refers to a genetic difference that causes a physiological deviation in a small number of individuals in a species compared to the majority of that species. The abnormality can be a positive abnormality or a negative abnormality. For example, a positive plant abnormality may be a genetic difference that makes some individual plants resistant to sunlight compared to other individuals in the species. Negative anomalies will make individuals in the same species of plants more susceptible to sun damage than normal plants.

  The method can best be described by referring to our survey on the genetic cause of Tangier disease. We began our investigation by probing a microarray containing nearly 60,000 normal human genes using RNA from cells cultured from individuals with Tangier disease, and we found that patients had almost zero The probe results could be used to identify ABC1 as a vascular gene in this single gene disease with high levels of circulating high density lipoprotein (HDL) and increased risk of heart disease. As a result of the vascular gene, there is no need to obtain a zero level detectable mRNA signal in such experiments. In this successful example, roughly 175 of the 58,800 probes on the microarray were underexpressed more than 2.5-fold in Tangier disease RNA-versus-normal. Several additional steps can be employed to confirm the identity of the culprit gene. They repeat such microarray probes in unrelated individuals with Tangier disease, determine the location of the chromosomal map of each gene, and compare it to the reported large gene intervals linked to the disease, candidate proteins and Includes consideration of similar functions of their homologs, biochemical tests, and sequencing of the best candidate genes in patients to find mutations. In these methods, gene expression microarray analysis can lead to the identification of genetic defects that have been genetically identified, such as the identification of ABC1 as a defect in Tangier disease.

  Further utility in this method is the disease sample vs. Other genes that are under- or over-expressed in normal include those that are differentially regulated as a result of genetic defects in the patient, either as a compensatory response or as a contributor to the cause of the disease. This can be used for drug development and can provide identification of other proteins in related biological pathways that can help elucidate the cause of the disease in relation to therapy and diagnosis. If gene deletion or other mutations cause complete absence of mRNA as observed in many examples of thalassemia (globin gene defect) and other genetic diseases, gene expression in disease-versus-normal samples Analysis can lead to the identification of lost genes in a more direct way.

  In these examples, the gene expression array probed with an RNA sample is of the type where the probe sample is cDNA aligned on a microscope slide, although other array techniques will suffice. These will include, but are not limited to, those that align DNA samples on filter membranes or use oligonucleotide probes synthesized on a “gene chip” by photolithography.

  In general, the term microarray refers to distinct oligonucleotides synthesized on a substrate such as paper, nylon or other type of membrane, filter, chip, cover glass, or any other suitable solid support. An array. Microarrays can be prepared, used and analyzed using methods known in the art (eg, Brennan, TM et al. (1995), each of which is incorporated herein by reference. US Pat. No. 5,474,796; PCT application WO 95-251116; Shalon, D. et al. (1995) PCT application WO 95/35505; and US Pat. No. 5,605,662).

  Array elements can be synthesized on the surface of the substrate using chemical coupling techniques and inkjet devices. Also, arrays similar to dots or slots, blots can be used to place and link elements to the surface of the substrate using thermal, UV, chemical or mechanical bonding techniques. A typical array can be created manually or by using available methods and machines, which can contain an appropriate number of elements. After hybridization, unhybridized probe can be removed and the level and pattern of fluorescence measured using a scanner. The degree of complementarity and relative abundance of each probe that hybridizes to elements on the microarray can be assessed through analysis of the scanned image.

  Full-length cDNAs, expressed sequence tags (ESTs), or fragments thereof can include microarray elements. Cross sections suitable for hybridization can be selected using software well known in the art, such as LASERGENE software (DNASTAR). Full-length cDNAs, ESTs or fragments thereof corresponding to the nucleotide sequences of abnormal individuals and normal individuals are placed on a suitable substrate such as a glass slide. The cDNA is fixed to the slide using, for example, UV crosslinking, followed by thermal and chemical treatment and subsequent drying (eg, Schena, M. et al. (1995) Science 270: 467-470; Shalon, D. et al. (1996) Genome Res. 6: 639-645). Probes such as fluorescent probes are prepared and used for hybridization to elements on the substrate.

  In order to perform sample analysis using a microarray, RNA or DNA from a biological sample is made into a hybridization probe. mRNA is isolated to yield cDNA, which is used as a template to make antisense RNA (aRNA). The aRNA is amplified in the presence of fluorescent nucleotides and the labeled probe is incubated with the microarray so that the probe sequence hybridizes to the complementary oligonucleotides of the microarray. Incubation conditions are adjusted so that hybridization occurs with exact complementarity matches or with varying degrees of lower complementarity. After removal of unhybridized probe, the level and pattern of fluorescence is measured using a scanner. The scanned image is examined to determine the degree of complementarity and relative abundance of each oligonucleotide on the microarray. A biological sample can be obtained from any bodily fluid (eg, blood, urine, saliva, mucus, gastric fluid, etc.), cultured cells, biopsies, or other tissue preparations. A detection system can be used to simultaneously measure the presence, absence, and amount of hybridization for all of the distinct sequences. This data can be used in large-scale correction experiments for sequences, mutations, variants or polymorphisms between samples.

  The microarray preferably consists of a large number of unique single stranded nucleic acid sequences, usually either synthetic antisense oligonucleotides or cDNA fragments bound to an individual support. The microarray can contain oligonucleotides covering known 5 'or 3' sequences, or sequential oligonucleotides covering the full length sequence; or selected from specific regions along the length of the sequence Unique oligonucleotides can be included. Polynucleotides for use in microarrays are one or more unidentified cDNAs that are specific for the gene or genes of interest for which at least a fragment of the sequence is known or that are common to a particular cell type, development or disease state It can be a specific oligonucleotide.

  In order to generate oligonucleotides to known sequences for microarrays, the gene of interest is examined using a computer algorithm starting at the 5 'or more preferably the 3' end of the nucleotide sequence. The algorithm identifies oligomers of a defined length that are unique to the gene, have a GC content within a range suitable for hybridization, and lack a predicted secondary structure that can interfere with hybridization. Oligomers are synthesized in designated areas on the substrate using a light-directed chemical process. The substrate can be paper, nylon or other type of membrane, filter, chip, glass slide or any other suitable solid support. Arrays can be made manually or using available devices (slot blot or dot blot apparatus) materials and machines (including robotic equipment), 8 dots, 24 dots, 96 dots, 384 dots, 1536 dots Or a grid of 6144 dots, or any other plurality of grids suitable for effective use of commercially available equipment.

  Once the genetic cause of the inherited anomaly has been fitted or identified, a potential or real defective portion of the gene can be used as a target in the microarray. Microarrays can be used to monitor the expression levels of a large number of genes and to develop and monitor potential therapeutic agents and the activity of therapeutic agents.

  The following examples further illustrate the invention, but should not be construed as limiting the invention in any way.

(Example 1)
This example shows that patients with Tangier disease (TD) have the absence of apoA-I-mediated lipid efflux.

Cell culture : Human fibroblasts were obtained from skin explants from two normal subjects (NL1) and three unrelated patients with Tangier disease (TD). TD1 cells were obtained from a 53 year old woman with extremely low plasma HDL cholesterol and apo AI levels and clinical signs typical of Tangier disease. TD2 cells were obtained from a 56 year old male with clinical, morphological and biochemical features of Tangier disease including very low levels of plasma HDL cholesterol and apo AI (Francis et al., J. Clin Invest., 96, 78-87 (1995)). TD3 cells were obtained from an 18-year-old male with Tangier's disease who presented with orange tonsils, asymmetric motor neuropathy, 5 mg / dL plasma HDL cholesterol and 16 mg / dL LDL cholesterol (Lawn et al., J. Clin. Invest., 104, R25-R31 (1999)). Normal cells and TD subject cells are described in Oram et al. Lipid Res. 40: 1769-1781 (1999). Briefly, cells were transfected with an amphotropic retrovirus containing human papillomavirus 16 oncodiyne E6 and E7 inserts and a vector with a neomycin resistance selection marker. Control cells were infected with the vector alone (mock infection). The pooled cell population was selected for 2 passages in the presence of G418, after which G418 was excluded from the medium. Fibroblasts were used between passages 5 and 16 (primary culture) or passages 6 and 15 (immortalization). Immortalized normal and TD cells are seeded into 16-mm wells or 35-mm dishes and grown to confluence in Dulbecco's modified Eagle's medium (DMEM) + 10% fetal bovine serum (FBS) before use in experiments I let you. RAW264.7 mouse monocytic cells (American Type Culture Collection, Rockville, MD) were also maintained in DMEM containing 10% FBS.

Assay to measure lipid efflux : ApoAI-mediated efflux of cholesterol and phospholipids is described in Francis et al. Clin. Invest. 96: 78-87 (1995). Cultured dermal fibroblasts from normal and TD subjects were cultured in the presence of 0.2-0.5 μCi / ml [ ³ H] cholesterol (40-60 Ci / mmol, Amersham Corp., Arlington Heights, IL). Labeled by growing to confluence. When the cells were 60-80% confluent, radioactive cholesterol was added to the serum-containing growth medium. After 3 days, the cells were washed twice with PBS / BSA, while simultaneously preventing growth and loading with cholesterol to maximize apolipoprotein-mediated lipid efflux. This is accomplished by incubating the cells for 48 hours in serum-free DMEM + 2 mg / ml fatty acid-free bovine serum albumin (DMEM / BSA) (Sigma Chemical Co., St. Louis, MO) and 30 μg / ml non-radioactive cholesterol. It was done. RAW264.7 cells were obtained from Smith et al., J. MoI. Biol. Chem. , 271: 30647-30655 (1996), and cholesterol-loaded through the scavenger receptor by incubation with acetylated LDL for 24 hours. Briefly, RAW264.7 cells in 24-well dishes were cholesterol-loaded and preincubated with AcLDL for 30 minutes at 37 ° C., 50 μl / ml 1 M glucose, 10 μl / ml 200 mM glutamine and 2% BSA. And in acetylated low density lipoprotein (AcLDL) and [ ³ H] -cholesterol supplemented overnight in 0.5 ml DMEM to give a final concentration of 0.33 μCi / ml [ ³ H] -cholesterol. Obtained. Subsequently, the cells were washed 5 times with PBS containing 1% BSA and incubated overnight (16-18 hours) in DMEM / BSA to equilibrate the cholesterol pool.

After equilibration of the cholesterol pool, cells were rinsed 4 times with PBS / BSA and incubated with DMEM / BSA for 1 hour at 37 ° C. prior to efflux incubation. Add effluent medium (DMEM / BSA) containing either albumin alone (control), albumin + HDL (40 μg protein / ml) or albumin + Apo AI (10 μg / ml, Biodesign International, Kennebunk, ME) Were incubated for 4, 24 or 48 hours. Phospholipids were labeled by including 10 μCi / ml [ ³ H] choline (75-85 Ci / mmol, Amersham Corp.) in DMEM / BSA overnight equilibration medium. Radioactivity found in the culture medium was measured by scintillation counting after 15 minutes centrifugation at 12,000 g. Radioactivity in the cells was solubilized in 0.5 ml of 0.2 M NaOH (Smith et al., J. Biol. Chem., 21: 30647-30655 (1996)) or Francis et al., J. Biol. Clin. Invest. 96, 78-87 (1995) as determined by scintillation counting after extraction in hexane: isopropanol (3: 2 v / v). Cells containing labeled phospholipids were extracted with 1 ml isopropanol for 1 hour and then with hexane: isopropanol as described above. Cholesterol or phospholipid efflux was expressed as the percentage of tritiated lipid count in the media over the total tritiated lipid count recovered from the cells and media (cpm media / cpm (medium + lysate) x 100).

As shown in FIGS. 1A and C, the addition of HDL or apoA-I results in the removal of cholesterol from cholesterol-loaded fibroblasts obtained from normal subjects. However, in TD cells, the concentration of HDL that removes cholesterol is slightly reduced and ApoA-I's ability to remove cholesterol is completely absent. FIG. 1 shows that normal and TD fibroblasts release approximately 3-4% [cells: ³ H] -cholesterol into the medium during 48 hours of incubation with albumin. Addition of HDL to albumin medium increases [ ³ H] -cholesterol efflux from both normal and TD fibroblasts, but to a lesser extent in TD cells (FIGS. 1B, D). Addition of apoA-I promoted [ ³ H] -cholesterol efflux from normal fibroblasts (FIGS. A and C), but against [ ³ H] -cholesterol efflux from TD fibroblasts Has little or no effect.

(Example 2)
This example shows that 175 genes show at least 2.5-fold decreased expression in TD cells and 375 genes show at least 2.5-fold increased expression compared to normal cells. Different gene expression was measured using gene-expression microarray (GEM) analysis of cDNA from normal individuals (non-TD) and from patients with TD.

Cell culture : Immortalized cell cultures obtained from normal individuals and TD patients described in Example 1 were used. Confluent cultures were maintained in DMEM / BSA and supplemented with 1 mM 8-bromo-ring adenosine monophosphate (8-Br-cAMP, Sigma Chemical Co., St. Louis, MO) for 24 hours.

mRNA extraction and cDNA synthesis : mRNA from both normal and TD fibroblasts was prepared from total RNA extracted from cells with Trizol (Life Technologies Inc., Bethesda, MD, Cat. # 15596-026). The mRNA was isolated using an Oligotex mRNA kit (Qiagen Inc., Valencia, CA, Cat. # 70022) according to the vendor's protocol. MRNA was reverse transcribed using Cy3 or Cy5 fluorescent dyes to obtain fluorescently labeled cDNA according to the method described in DeRisi et al., Science, 24: 680-686 (1997). CDNA obtained from TD cells was labeled with Cy3 fluorescent dye, and cDNA from normal cells was labeled with Cy5 fluorescent dye (Incyte Genetics, Palo Alto, Calif.).

Microarray analysis : To analyze different gene expression in cells from individuals with TD and normal individuals, Cy3 and Cy5 fluorescently labeled cDNA samples prepared as described above were analyzed on a microscope slide (Incyte Genomics, Palo Alto, Calif.). Hybridized to a set of Gene Album microarrays (GEM). Each of the six slides contained approximately 9,800 human cDNA samples + 200 control samples, resulting in a 58,800 partial cDNA microarray. Therefore, an estimate of redundancy was made, representing approximately 30-50% of the expressed human genes. Hybridization of Cy3-labeled cDNA prepared from TD1 cells and Cy5-labeled cDNA from normal cells was performed using TD cells vs. Comparison of the relative RNA content of normal cells for the expressed gene was made possible. In addition, Cy3-labeled cDNA prepared from TD2 cells and Cy5-labeled cDNA from normal cells were hybridized to the same set of microarrays to examine gene expression variation between different TD patients.

Results : Data were analyzed using GemTools software (Incyte Genotics, Palo Alto, Calif.) And expressed as the ratio of TD cell mRNA to normal cells. The results showed that most genes are relatively expressed in TD1 and normal cells. As shown in FIG. 2 (left side of the diagonal line in the previous section), only 175 genes are under-expressed 2.5 times in TD1 cells compared to normal cells, whereas 375 genes are expressed in TD1 cells compared to normal cells. Genes that are more highly expressed in TD cells overexpressed 2.5 times (lower and to the right of the diagonal) differ as a result of tangier mutations as a compensatory response or as a contribution to disease pathology Including what is adjusted. Genes that contribute to the observed phenotype of TD and are more highly expressed in TD cells are interferon-β (IFN-β), macrophage inflammatory protein-2α, granulocyte chemotactic protein-2, IL- 11, including prostaglandin endoperoxide synthase-2 (COX-2), thrombospondin and monocyte chemotactic proteins 1, 3 and 4 (Lawn et al., J. Clin. Invest., 104: R25-R31 ( 1999)).

  No single RNA expressed in normal fibroblasts was found that was completely absent in either TD1 or TD2 cells. Also, comparison of differentially expressed genes in TD1 and TD2 revealed that there was little variation between individual TD patients. For example, of the most highly down-regulated genes in TD2 cells, 92% were also underexpressed in TD1 cells compared to normal cells. Of the genes, TD1 or TD2 vs. It was the gene for ABC1 protein that was 2.5-fold underexpressed in normal cells. The ABC1 gene was tracked due to some attributed function of its homologue, and because the gene was located approximately in the chromosomal region reported as the TD gene region.

(Example 3)
This example shows that the ABC1 gene is located on human chromosome 9q31.

Previous gene linkage analysis mapped the TD gene to the 7-cM region of human chromosome 9q31 (Rust et al., Nat. Genet., 20, 96-98 (1998)). In addition, in situ hybridization analysis revealed that the ABC1 gene is located at a wider chromosomal spacing 9q22-9q31 (Luciani et al., Genomics, 21, 150-159 (1994)). Using the PCR method in the GeneBridge4 panel of a human / hamster irradiated hybrid (Research Genetics, Inc., Huntsville, AL), human ABC1 was determined to be located between the markers WI-14706 and WI-4062. This corresponds to the 7-cM region of 9q31. The DNA from the 93 human / hamster hybrid cell line is human ABC1-specific primer LF:
CCTCTCATTACACAAAAAACCAGAC (SEQ ID NO: 11) and LR:
GCTTTCTTTCACTTCTCATCCTG (SEQ ID NO: 12)
Amplified by PCR using Each system was scored as positive or negative for the human ABC1 amplification product and the ABC1 mapping obtained from the analysis of this data is the Internet (http://carbon.wi.mit.edu:8000/cgi-bin/contig) /Fhmapper.pl) was achieved using the Whitehead Institute / MIT Center for Genome Research Software. These results were further confirmed by Southern blot hybridization to human genome / yeast artificial chromosome clones (Research Genetics, Inc.) from comparable intervals. In addition, public database searching (GeneMap '98; National Center for Biotechnology Information) and irradiation hybrid mapping eliminated other significantly underexpressed genes in microarray data from positions in reported gene intervals. These supplementary data indicate that the ABC1 gene is located on human chromosome 9q31, and further indicate that the ABC1 gene is associated with Tangier disease.

(Example 4)
This example shows the determination of the nucleotide sequence of the wild type ABC1 gene including the flanking region and the entire coding region.

  DNA sequencing was performed using an ABI Prism 310 gene analyzer or by Davis sequencing (Davis, CA). Both strands were sequenced in their entirety. The sequence of the ABC1 gene open reading frame from normal subjects was determined from a full-length cDNA clone obtained from an expression plasmid library constructed from normal fibroblast RNA. CDNA was synthesized according to the Stratagene kit protocol (Stratagene, La, Jolla, Calif.) To construct the plasmid library. Briefly, first strand cDNA was synthesized from mRNA using oligo-bT primer with XhoI site and MMLV reverse transcription structure in the presence of 5-methyl cCTP. The second strand was synthesized using RNase H and DNA polymerase I in the presence of unmodified dLTP. After making the cDNA blunt with pfu DNA polymerase, an EcoRI linker was ligated to the cDNA. The cDNA was then digested with XhoI to generate a XhoI end at the 3 'end of the cDNA. During the first strand synthesis, the internal XhoI site was protected from this digestion by semi-methylation. The synthesized cDNA was cloned into the HindIII and XhoI sites of the expression vector containing plasmid pCEP4 (Invitrogen Corp., Carlsbad, CA # VO44-50), cytomegalovirus promoter / enhancer. A 585 bp ABC1 probe was obtained by reverse transcription structure polymerase chain reaction (RT-PCR) using primers based on the known ABC1 sequence which was 5'-TCCTTGGGTTCAGGGGATTATC (SEQ ID NO: 13) and 5'-CAATGTTTTTGTGGCTTCGCC (SEQ ID NO: 14). Created. Using this ABC1 probe, clones containing a 10.5 kb insert of human ABC1 cDNA were recovered from the library using the CloneCapture selection kit (CLONTECH Laboratories, Inc., Palo Alto, Calif.) According to the manufacturer's protocol. This clone is shown in FIG. 3 as pCEPhABC1. The 10.5 kb ABC1 cDNA insert sequence is shown in SEQ ID NO: 1. By sequencing, pCEPhABC1 contains a human ABC1 open reading frame +5 'and 3' untranslated region of 6783 nucleotides, see Langmann et al. In Biochem. Biophys. Res. Comm. 257, 29-33 (1999) (GenBank accession number AJO12376), which was confirmed to have a larger open reading frame than the cDNA sequence reported.

(Example 5)
This example shows the sequence differences between the wild type ABC1 gene and the TD1, TD2 and TD3 gene sequences.

CDNA synthesis of TD1, TD2 and TD3 : cDNA is (1) sacIhabcf, 5′-AGTCGAGCTCCAAACATGTCAGCCTGTTACTGGAAGTGGGCC (SEQ ID NO: 15);
habcr3581, 5′-TCTCTGGATTCTGGGTCCTATGTCAG (SEQ ID NO: 16) and (2) habccf3585, 5′-GGGAGCTCTTTGTGGAACTCTTTC (SEQ ID NO: 17); habcrsalI, 5 ′
-Manufacturer's protocol (CLONTECH, Palo Alto, CA; Cat. Prepared from TD1, TD2 and TD3 cells by reverse transcription polymerase chain reaction (RT-PCR) using the Suerscript Choice cDNA system and Advantage cDNA polymerase mix. Amplification of 0.2-0.5 μg poly A + RNA with these primers at a final concentration of 0.4 μM resulted in two overlapping templates of approximately 3.5 kb. The template was gel-purified using the QIAEX II system (QIAGEN, Inc., Valencia, CA; Cat. # 20021) and adjusted to a concentration of 100 ng / μl.

Sequencing of TD1, TD2, and TD3 cDNAs : Sequencing of 8 μl of each template created as described above in a reaction with individual sequencing primers designed based on the wild-type ABC1 sequence at a final concentration of 0.5 μM. did. The primers were as follows: 1F: 5′-TTTCCTGGTGGACAATGAA (SEQ ID NO: 19), 2F: 5 ′
-AGTGACATGCGACAGGAG (SEQ ID NO: 20); 3F: 5'-GATCTGGAAGGCATTGGG (SEQ ID NO: 21); 4F: 5'-CCAGGCAGCATTGAGCTG (SEQ ID NO: 22); 5F: 5'-GGCCTGGACAACAGCATA (SEQ ID NO: 23); 5′-GGACAACCCTTTTGAGAGT (SEQ ID NO: 24); 7F: 5′-AAGACGACCACCCATGTCA (SEQ ID NO: 25); 8F: 5′-ATATGGGAGCTGCTGTCTG (SEQ ID NO: 26); 10F: 5′-AAGAGACTGCCTATATGCC (SEQ ID NO: 28): 11F: 5′-AGCGACAAAATCAAGAAG (SEQ ID NO: 29); 12F: 5'-TGGGCATGCAATCAGCTCT (SEQ ID NO: 30); 13F: 5'-TCCTCCACCAATCTGCCT (SEQ ID NO: 31); 14F: 5'-TTCTTCCTCATTACTGTT (SEQ ID NO: 32); 15F: 5'-GATCGCCATCAGACGTG No: 33); 16F: 5′-AGTGTCCCAGCATCTAAA (SEQ ID NO: 34); 1R: 5′-CAAAGTTCACAAATACTT (SEQ ID NO: 35); 2R: 5′-CTTAGGGCACAATTTCCACA (SEQ ID NO: 36); 3R: 5′-TGAAGTTGATGAT (SEQ ID NO: 37); 4R: 5′-TTTTTCACCATGTCGATGA (SEQ ID NO: 38); 5R: 5′-CTCCACTGATGGAACTGC SEQ ID NO: 39); 6R: 5'-GTTTCTTCATTTGTTTGA (SEQ ID NO: 40); 7R: 5'-AGGGCGGTTCTGGGATTG (SEQ ID NO: 41); 8R: 5'-CAGAATCATTGGATCAG (SEQ ID NO: 42); 9R: 5'- CATCAGAACTGCTCTGAG (SEQ ID NO: 43); 10R: 5′-AGCTGGCTTGTTTGCTTT SEQ ID NO: 44), 11R: 5′-TGGAACGCCCCAGCTTCACA (SEQ ID NO: 45), 12R: 5′-CCTGCCCATGCCACACACA (SEQ ID NO: 46), 13R -CTCATCACCCCGCAGAAAG (SEQ ID NO: 47), 14R: 5'-CACACTCCATGAAGCGAC (SEQ ID NO: 48), 15R: 5'-TCCAGATAATGC GGGAAA (SEQ ID NO: 49), 16R: 5′-TCAGGAGTGGCTTCAGGA (SEQ ID NO: 50), UTR1R: 5′-AAGTTGAGCTGGATTTCTG (SEQ ID NO: 51).

Results : Nucleotide numbering follows the numbering found in Lawn et al. (1999). Patient TD1 carries a full length open reading frame and has two substantial differences from the wild type sequence (SEQ ID NO: 8). One of these is an A to G substitution resulting in a glutamine to arginine residue change at position 537 of the 2201 amino acid sequence, as published by Lawn et al. (1999). The position of this residue is in the NH2-terminal hydrophilic domain, near the first predicted transmembrane domain. Patient TD2 also possesses an open reading frame with a substitution of arginine to tryptophan at residue 527 (SEQ ID NO: 10). Thus, both TD1 and TD2 contain substitutions that alter the charge of amino acids in the same region of the protein. TD3 DNA contains a 14 nucleotide insert in its ABC1 cDNA following nucleotide 5697 in one allele and a 138 bp insert after nucleotide 5062 in the other allele.

  Genomic sequencing of TD1, TD2 and TD3 DNA confirmed the changes found in each cDNA. Genomic sequences were created by PCR amplification of a 156 bp region of genomic DNA isolated from fibroblasts containing mutations found in cDNA from TD1 and TD2. Genomic sequencing also showed that patient TD1 was homozygous for glutamine to arginine substitution. Genomic DNA analysis showed that TD2 was compound heterozygous for one allele containing the detected substitution and a second allele containing an undetermined defect (not producing detectable mRNA). No substitution mutations were found in over 80 alleles of genomic DNA of non-TD individuals. The TD3 insertion was identified by sequence analysis and confirmed by RT-PCR using primers surrounding the insertion point. The 14-bp insertion following nucleotide 5697 caused a frameshift, resulting in a substitution of the wild type amino acid sequence from a position before the second ATP binding domain to the point of immature protein termination. The 138 bp insertion following nucleotide 5062 in the other allele contains an in-frame stop codon.

(Example 6)
This example shows that inhibitors of ABC1 transport activity inhibit apo AI-mediated cholesterol efflux from fibroblasts.

To test whether inhibition of ABC1 could affect the process of apolipoprotein-mediated cholesterol efflux, two compounds reported to be ABC1 inhibitors were assayed to monitor apolipoprotein-mediated cholesterol efflux. did. Compounds 4, 4-diisothiocyanostilbene-2, 2'-disulfonic acid (DIDS) and sulfobromophthalein (BSP) have been reported to inhibit the anion transport activity of ABC1 in a dose-dependent manner (Becq et al. , J. Biol. Chem., 272: 2695-2699 (1997); Hamon et al., Blood, 90: 2911-2915 (1997)). The apolipoprotein-mediated cholesterol efflux assay was performed as described in Example 1 with the indicated changes. Cholesterol-loaded and [ ³ H] cholesterol-labeled normal fibroblasts (n = 3) were incubated for 6 hours with or without 5 μg / ml apoA-I and 0, 0.2 mM or 0.4 mM DIDS. In addition, cholesterol-loaded and [ ³ H] cholesterol-labeled normal fibroblasts (n = 3) were incubated for 6 hours with or without 5 μg / ml apo AI and 0, 0.2 mM or 0.4 mM BSP. [ ³ H] cholesterol efflux was measured by scintillation counting as described in Example 1 and calculated as the percentage of total radiolabeled cholesterol appearing in the medium. Results are shown in FIG. 5 as the mean ± SD (n = 3) of efflux in the presence of ApoA-I after subtracting the value for ApoA-I-free medium. FIG. 5 shows that both DIDS and BSP inhibit the 6 hour efflux of tritiated cholesterol mediated by apolipoprotein AI. In addition, similar inhibition was observed in tritiated phosphatidylcholine efflux using DIDS and BSP (data not shown). The results of these tests are similar to the outflow defects in fibroblasts from patients with TD described in Example 1.

(Example 7)
This example demonstrates that antisense inhibition of ABC1 mRNA expression inhibits apo AI-mediated cholesterol efflux from fibroblasts.

Normal skin fibroblasts were labeled with [ ³ H] cholesterol as described in Example 1. 30 μM control morpholino oligonucleotide (5′-CCTCTTACCTCAGTTACAATTTTATA-3 ′ corresponding to the antisense complement of β-globin thalassemia mRNA; SEQ ID NO: 52) or 30 μM ABC1 antisense morpholino oligonucleotide (5′-CATGTTTCATTAGGGTGGGTCGC-3 SEQ ID NO: 53) Cells were loaded with oligonucleotides by scraping in the presence of either and replating on a new dish. Control cells were mock-loaded after [ ³ H] cholesterol-labeling by scraping in the absence of oligonucleotide and re-inoculation. Apo AI-mediated efflux was measured after 12 hours by scintillation counting as a percentage of total radiolabeled cholesterol appearing in the medium. Results are shown in FIG. 6 as the mean ± SEM of three independent experiments, normalized to the value for apoA-I-specific efflux in the absence of oligonucleotide in each experiment. As shown in FIG. 6, the antisense oligonucleotide directed against ABC1 mRNA is 50% of the cholesterol efflux from normal fibroblasts compared to the control antisense oligonucleotide (β-globin antisense oligonucleotide). Caused a decline.

(Example 8)
This example shows that overexpression of the human ABC1 gene results in increased apo AI-mediated cholesterol efflux from monocytes.

Stable transfection of RAW264.7 cells : Mouse monocyte RAW264.7 cells were stably transfected with the pCEPhABC1 expression plasmid for human ABC1. The construction of the pCEPhABC1 plasmid containing the open reading frame of human ABC1 is described in Example 4. Approximately 1 × 10 ⁶ RAW 264.7 cells with 2 μg p in 0.8 ml serum-free DMEM with CEPhABC1 DNA and 12 μl of gENEPROTER transfection reagent (Gene Theraphy Systems, Inc., San Diego, CA; CAT. # T201007). Was transfected for 5 hours. After 2 days, the cells were split in a ratio ranging from 1: 2-1: 50 and selection was applied by adding 150 μg / ml hygromycin to the culture medium. Two weeks later, hygromycin-resistant colonies were picked and expanded.

ApoA-I-mediated cholesterol efflux assay : Parent RAW264.7 cells and three clonal lines (L3, L5 and L6) stably expressing human ABC1 were grown to confluence. Cells were cholesterol-loaded and labeled by incubation with 0.5 μCi (ml) [ ³ H] cholesterol and 50 μg / ml acetylated LDL as described in Example 1. After equilibration of the cholesterol pool by overnight incubation in DMEM / BSA, the cells were washed and effluent medium was added as described in Example 1. ApoA-I-mediated cholesterol efflux was measured as described above by scintillation counting of tritiated cholesterol in cell medium and expressed as a percentage of the total count recovered from the cells and medium. Results are expressed as the mean ± SEM of 3 separate experiments normalized to the value for apoA-I-specific efflux from parental RAW264.7 cells within each experiment. FIG. 7 shows apoA-I-mediated cholesterol efflux from parental RAW264.7 cells and L3, L5 and L6 transfected cell lines. As will be appreciated, transfection with the ABC1 expression vector results in a 4-fold (L6) to 6-fold (L3 and L5) increase in apoA-I-mediated cholesterol efflux. These results indicate that overexpression of the ABC1 gene can substantially increase the amount of cholesterol efflux from macrophage cells.

Example 9
This example shows that ABC1 mRNA expression is regulated in normal skin fibroblasts by a cellular state associated with cholesterol efflux, but not in TD fibroblasts.

  To determine whether ABC1 plays a rate-limiting role in cellular sterol efflux, ABC1 synthesis was measured under various cellular conditions associated with the cholesterol efflux process. Specifically, normal fibroblasts and TD fibroblasts were individually exposed to excess cAMP, cholesterol or apo AI conditions. Cell cultures of normal skin fibroblasts and TD1 and TD2 fibroblasts were prepared as described in Example 1. ABC1 mRNA levels were measured by RT-PCR.

Cell culture : Immortalized cell cultures of normal skin fibroblasts and TD1 and TD2 fibroblasts were prepared as described in Example 1. Cells were grown to confluence for 24 or 48 hours in DMEM / 10% FBS prior to replacement with DMEM / BSA and indicated additives. RNA was prepared as described in Example 2.

RT-PCR : Quantitative PCR was performed using GeneAmp 5700 Sequence Detection System (Perkin-Elmer Applied Biosystems, Foster City, Calif.). Briefly, 500 ng DNase-treated mRNA was reverse transcribed using 2.5 μ random hexamer primers. SYBR Green Core Kit (PE Applied Biosystems, Foster City, CA; # 4304886) and human ABC1 primer LF: 5′-CCTCTCATTACAAAAAACCAGAC (SEQ ID NO: 11) and LR: 5′-GCTTTCTTTCACTTCCT12 Almost 5% of this reaction was amplified to obtain an 82 bp fragment corresponding to nucleotides 7018-7099 of human ABC1. PCR cycle conditions were as follows: 10 minutes at 95 ° C; followed by 95 ° C, 40 seconds for 15 seconds and 60 ° C for 60 seconds. MRNA in each sample was quantified by detecting the increase in fluorescence caused by SYBR green binding to the double stranded amplification product that occurred during each PCR cycle. All samples were run in triplicate, normalized to β-actin mRNA, and with primers actin F: 5′-TCACCCACACTGTGCCATCTACGA (SEQ ID NO: 54) and actin B: 5′-CAGCGGGAACCGCTCCATTGCCAATGG (SEQ ID NO: 55) Amplified in parallel reaction. Standard curves were generated for both ABC1 and β-actin on the same PCR plate.

8-Br-cAMP assay : Normal, TD1 and TD2 fibroblasts were grown to confluence in DMEM / 10% FBS and then treated with 1 mM 8-Br-cAMP in DMEM / BSA for 24 hours.

Cholesterol assay : Normal, TD1 and TD2 fibroblasts were grown to confluence in DMEM / 10% FBS and then treated with 30βg / ml free cholesterol in DMEM / BSA for 48 hours followed by DMEM / BSA Equilibrated for 18-24 hours.

Apo AI assay : Normal, TD1 and TD2 fibroblasts were grown to confluence in DMEM / 10% FBS and then treated with 30 μg / ml free cholesterol in DMEM / BSA for 48 hours, followed by DMEM / Equilibrated in BSA + 10 μ / ml Apo AI for 18-24 hours.

Results : FIG. 8 shows that in normal fibroblasts, ABC1 mRNA is increased approximately 10-fold by exposure to 8-Br-cAMP and approximately 17-fold by exposure to cholesterol in serum-free medium. Subsequent exposure of cholesterol-loaded cells to Apo AI results in a marked decrease in ABC1 mRNA expression. Although no mechanism has been shown, previous studies have shown that cholesterol efflux is promoted in the presence of compounds such as cAMP and cholesterol (Hokland et al., J. Biol. Chem., 268: 25343-25349). (1993)). The results show that in normal fibroblasts, ABC1 mRNA is induced by these known effectors of the cholesterol efflux pathway and is suppressed by exposure to the apolipoprotein cholesterol receptor, which indicates that ABC1 expression is 4 shows that apolipoprotein-mediated regulation by cellular conditions associated with cholesterol efflux. In contrast, fibroblasts from TD patients are not prepared by cholesterol efflux effectors. First, the cAMP-induced level of ABC1 mRNA in both TD1 and TD2 cells is only about 40% that of normal cells. Furthermore, exposure of cholesterol-loaded cells to apoA-I did not alter ABC1 expression (TD1 cells) or slightly increased ABC1 expression (TD2 cells). These results reflect a defect in apo-AI mediated cholesterol efflux described for TD cells. Interestingly, cell growth in serum-containing medium suppressed ABC1 message to near the detection limit (data not shown). This may reflect the fact that the function of the lipid efflux pathway requires cell arrest or other cellular conditions that require reduced cholesterol. In conclusion, conditions associated with increased efflux of cellular cholesterol (ie, cholesterol loading, cAMP treatment, serum deprivation) result in increased expression of ABC1 mRNA in normal fibroblasts. Conversely, exposure of cholesterol-loaded normal fibroblasts to apoA-1 reduces ABC1 expression.

(Example 10)
This example shows that a ligand for the LXR nuclear receptor such as 20-hydroxycholesterol, and a ligand for the RXR receptor such as 9-cis retinoic acid can increase ABC1 gene expression in mouse RAW264.7 cells. .

  The LXR nuclear receptor is a transcription factor that forms an absolute heterodimer with the nuclear receptor RXR, which is activated by binding to a class of oxysterols including 22-hydroxycholesterol and 20-hydroxycholesterol. Enhance the transcription of its target gene (Janowski et al., Nature, 383: 728-731 (1996)). As such, they are candidates for mediating cholesterol-induced gene transcription. Furthermore, experiments have shown that ABC1 mRNA and protein are increased in fibroblasts and macrophages in response to cholesterol loading, and that LXR and RXR expression is increased in cholesterol-loaded macrophage cells by exposure to oxidized LDL. LXR and RXR nuclear reporters are quite potential candidates for the transcriptional activator of the ABC1 gene. To determine whether the LXR and RXR reporters play a role in ABC1 gene expression, the level of ABC1 mRNA was measured in response to 20-hydroxycholesterol and 9-cis retinoic acid.

  Mouse RAW 264.7 cells are grown to confluence in DMEM / 10% FBS and then contain 9-cis retinoic acid (10 μM), 20-hydroxycholesterol (10 μM) or a combined ligand (20 μM total) Treated in serum-free DMEM / BSA for 24 hours. Control cells received only ethanol vehicle (0.1% v / v). RNA was extracted and treated with DNase and ABC1 mRNA was measured by RT-PCR using PE Biosystems SYBR Green Technology as described in Example 9. FIG. 9 shows that ABC1 mRNA expression is increased as a result of treatment with either 20-hydroxycholesterol or 9-cis retinoic acid. In addition, Example 9 shows that treatment with both ligands together resulted in a significant synergistic effect, almost a 6-fold increase over ABC1 expression observed with either ligand alone. . These results indicate that ligands for the nuclear receptors LXR and RXR can increase the expression of the ABC1 gene.

(Example 11)
This example shows that enhanced expression of ABC1 protein in the plasma membrane is associated with lipid efflux.

  Cell-surface labeling and immunoprecipitation were used to determine whether increased expression of ABC1 protein in the plasma membrane correlates with increased cholesterol efflux (FIG. 10). The relative amount of ABC1 on the cell surface is determined by cross-linking surface proteins on intact cells with the membrane impermeant sulfo-NHS-biotin, followed by membrane solubilization, immunoprecipitation with ABC1 antibody, SDS-PAGE and streptometry. Measured by process with detection with avidin.

Cell culture : Normal and TD1 fibroblasts were immortalized as described in Example 1. Both normal and TD1 cells were cultured under controlled conditions and conditions known to increase apolipoprotein-mediated cholesterol efflux (Oram. Et al., J. Lip. Res., 40: 1769-1781 (1999)). . Control cells were grown to confluence in DMEM / 10% FBS and then incubated for 18 hours in DMEM / BSA without any additives (control). cAMP-treated cells were grown to confluence in DMEM / 10% FBS and then incubated for 18 hours in DMEM / BSA containing 1 mM 8-Br-cAMP (cAMP). Cholesterol-loaded cells were grown to confluence in DMEM / 10% FBS and then incubated in DMEM / BSA containing 30 μg / ml cholesterol for 48 hours and 18 hours without additives (cholesterol). CAMP-treated cholesterol-loaded cells are grown to confluence in DMEM / 10%, then 48 hours in DMEM / BSA containing 30 μg / ml cholesterol, and 18 hours because of containing 1 mM 8-Br-cAMP. Incubated (cholesterol + cAMP).

Cell-surface labeling : for selective labeling of plasma membrane ABC1 at 9 ° C. containing 1 mg / ml sulfo-nhS-biotin (containing 30 μg / ml cholesterol (Pierce, Rockford. IL; Cat. # 21217) See biotinylated for 30 minutes (Walker et al., Biochemistry, 50: 14009-14014 (1993)).

Immunoprecipitation : Rabbit antiserum for ABC1 was raised against a synthetic peptide corresponding to the putative peptide KNQTVVDAVLTSFLQDEKVKES located at the C-terminus of human ABC1. By solubilizing the cells in PBS containing 1% Triton X-100 (Sigma, St. Louis, MO) and the protease inhibitor leupeptin (1 mM), and pepstatin (1 mM) and aprotinin (1 mM) Immunoprecipitation was performed. The cell extract was incubated with anti-ABC1 antiserum at 1: 200 dilution overnight at 4 ° C. and 5 μl of protein A-coated magnetic beads (Dynal, Lake Success, NY; Cat. # 1001. 01) for another hour. The antibody-antigen complex was precipitated with a magnet, the beads were washed twice with 1% Triton-X / PBS, and the protein was eluted with 1% acetic acid.

Detection of ABC1 protein : The eluted biotinylated protein was subjected to SDS-PAGE (6% gel; 150 V, 5 hours) and transferred to a nitrocellulose membrane (200 mA, 18 hours). The nitrocellulose was probed with streptavidin-horseradish peroxidase (Amersham Pharmacia Piscataway, NJ; Cat. # RPN1231), diluted 300-fold and augmented according to vendor protocol (Amaersham Piscataway, Piscataway, NJ) Detected by label (ECL). To test for possible biotinylation of intracellular proteins, the supernatant after immunoprecipitation was treated with a mouse monoclonal antibody against intracellular protein β-COP, and the immunoprecipitated biotinylated β-COP was assayed by streptavidin blotting. Nothing was detected.

Result : As shown in FIG. 10, the 240 kDa ABC1 protein appears as a doublet. In both normal (10A) and TD1 (10B), ABC1 protein is partially localized to the plasma membrane. Similar results were observed with the second normal fibroblast line and with TD2 fibroblasts (data not shown). The cell-surface expression of ABC1 was slightly increased when cells grown in serum (normal and TD1 cells) were treated with 8-Br-cAMP. Serum depletion and cholesterol-loading of both normal and TD1 cells markedly increased the cell-surface expression of ABC1, which was further enhanced by cAMP treatment. These results indicate that the expression of ABC1 on the cell surface is regulated by conditions that enhance apolipoprotein-mediated lipid efflux, which is that its localization to the plasma membrane plays a key role in its lipid transport function Show that it fits the idea. Mutations in TD1 and TD2 cells do not appear to severely impair ABC1 expression or processing, which may have secondary effects on lipid transport or interaction with accessory proteins, where the mutation is It depends on what happens.

Example 12
This example shows that agents that inhibit the degradation of 3 ', 5' cyclic AMP, such as phosphodiesterase inhibitors, increase apolipoprotein AI-mediated efflux from macrophage cells.

As shown in FIG. 10, cAMP increases the activity of ABC1. This experiment was performed to measure cholesterol efflux from macrophage cells in the presence of elevated cAMP. Elevated levels of cAMP can be achieved in the presence of agents that stimulate cAMP synthesis or inhibit cAMP degradation. For example, rolipram is a compound that modulates cAMP levels by inhibiting phosphodiesterases (a group of enzymes that degrade cAMP). The effect of elevated cAMP on cholesterol efflux was measured using the apolipoprotein-mediated cholesterol efflux assay described in Example 1. Briefly, RAW264.7 cells suspended at a density of 1.25 × 10 ⁵ cells / ml were grown in DMEM / 10% FBS supplemented with pyruvate. After 24 hours, the medium was removed and replaced with DMEM / BSA + radiolabeled cholesterol (1 μCi / ml [ ³ H] -cholesterol) and 50 μg / ml acetylated LDL at 24 hours. Next, in an equilibrium medium consisting of either DMEM / BSA + ApoA-I alone (20 μg / ml), ApoA-I and 8-bromo 3 ′, 5 ′ cAMP (1 mM) or ApoA-I and rolipram (50 μM) Cells were maintained for 24 hours. After 12-24 hours, [ ³ H] cholesterol efflux was measured by scintillation counting as described in Example 1 and calculated as the percentage of total radiolabeled cholesterol appearing in the medium. The results showed that cholesterol-loaded control cells that did not take ApoA-I showed 3% cholesterol efflux, whereas cells that took ApoA-I alone showed 5% efflux. Cholesterol-loaded cells ingesting apo AI and cAMP show 32% cholesterol efflux, indicating that elevated cAMP promotes cholesterol efflux. Similarly, cells ingesting apo AI and phosphodiesterase inhibitor (rolipram) showed 17% cholesterol efflux.

(Example 13)
This example shows that agents that are ligands for nuclear receptors such as LXR, RXR and PRAR nuclear receptors increase apolipoprotein AI-mediated efflux from macrophage cells.

  To determine whether ligands for nuclear receptors affect apolipoprotein-mediated cholesterol efflux, various ligands were tested using the apoA-I-mediated cholesterol efflux assay described in Example 12. The nuclear receptor superfamily has been implicated in lipid metabolism (Russell. DW, Cell, 97: 539-542 (1999); Spiegelman, BW, Cell, 93: 153-155 (1998). Janowski et al., Nature, 383: 728-731 (1996)) Includes several members such as the liver receptor LXR, the retinoid receptor RXR and the peroxisome proliferator-mediated receptor PPAR. In addition, ligands for some of these receptors have been observed to increase plasma HDL, and gene expression profiling (microarray) data respond to cholesterol load through LDL with the participation of hormone receptors. showed that. Using the assay described above, 9 cis-retinoic acid (RXR ligand), oxysterol (LXR ligand) and fenfibrate (PPAR ligand) were tested to determine the effect on cholesterol efflux. Cholesterol-loaded control cells that did not take apoA-I showed 3% cholesterol efflux, while cells that took only apoA-I showed 5% efflux. In contrast, cholesterol-loaded cells ingested with Apo AI and 9 cis-retinoic acid (30 ng / ml) showed 16% cholesterol efflux. Cells receiving apo AI and oxysterol (5 μg / ml) showed 14% cholesterol efflux. Cells ingesting Apo AI and fenfibrate (3 μg / ml) showed 10% cholesterol efflux. These results indicate that hormone receptors can be modulated to increase the rate of apolipoprotein-mediated cholesterol efflux from macrophages.

  Furthermore, when efflux assays were performed using various concentrations of 9-cis-RA (0.3 ng / ml, 3.0 ng / ml or 30 ng / ml), the results show that 9-cis-RA is dose dependent. It has been shown to mediate cholesterol efflux from macrophages. Specifically, control cells (Apo A-I only) were obtained from 1890c. p. m. 0.3 ng / ml 9-cis-RA was 1522 c. p. m. 3.0 ng / ml 9-cis-RA was 3568 c. p. m. And 30 ng / ml 9-cis-RA was 8597 c. p. m. showed that. In addition, using a similar assay in which RAW264.7 cells are cholesterol-loaded for 48 hours, other nuclear receptor activators such as 22-hydroxycholesterol (LXR ligand) and benzfibrate have been shown to increase cholesterol efflux. (Data not shown).

(Example 14)
This example shows that eicosanoids such as prostaglandin E1 and prostaglandin PG12 increase apolipoprotein AI-mediated efflux from macrophage cells.

  Eicosanoids such as prostaglandins and prostacyclins have been shown to be effective in the treatment of hypercholesterolemia. To determine whether eicosanoids affect apolipoprotein-mediated efflux, PEG1 and PG12 were tested using the apo AI-mediated cholesterol efflux assay described in Example 12. This assay showed that cholesterol-loaded control cells that received ApoA-I had 3% cholesterol efflux and cells that received ApoA-I alone had 5% efflux. Cholesterol-loaded cells ingesting Apo AI and PG12 (25 nM) showed 10% cholesterol efflux. Cells ingesting Apo AI and PEG1 (25 nM) showed 15% cholesterol efflux. These results indicate that eicosanoids can increase the rate of apolipoprotein-mediated cholesterol efflux from macrophages.

(Example 15)
This example demonstrates that compounds can be tested for their ability to modulate ABC1 gene expression using a reporter gene under the control of the ABC1 promoter.

  A recombinant plasmid was created using the pGL3 luciferase reporter vector system (Promega, Madison, WI), and the expression of the reporter gene under the control of the ABC1 promoter was measured.

Construction of reporter plasmid : Plasmid pGL3-Basic (Pronega, Madison, Wi; Cat. # E1751) was used as a control plasmid containing a luciferase gene without a promoter. A reporter containing the ABC1 promoter and the luciferase gene is a genomic fragment from the 5 ′ flanking region of the ABC1 gene (hAPR1 5 ′ promoter, corresponding to nucleotides 1080-1643 of SEQ ID NO: 3) in the SacI site of the GL3-Basic plasmid. It was made by cloning to obtain plasmid GL-6a. The plasmid GL-6a was then digested with SpeI and Acc65I . The BsiWI - SpeI fragment excised from the lambda subclone, representing the ABC1 genomic sequence corresponding to nucleotides 1-1542 of SEQ ID NO: 3, was ligated to the remaining vector: ABC1 promoter generated by this digestion. The resulting plasmid pAPR1 encodes a luciferase reporter gene under the 1.75 kb transcriptional control of the human ABC1 promoter sequence.

Reporter construct transfection : The control or pAPR1 plasmid was transfected into a confluent culture of RAW246.7 cells maintained in DMEM containing 10% fetal bovine serum. Each well of a 12-well dish is either pGL3-Basic, pGL3-promoter or pAPR1 DNA (1 μg), luciferase plasmid DNA (1 μg) and 12 μl Geneorder reagent (Gene Therapy Systems, San Diego, Calif .; Cat. # T201007) And transfected for 5 hours. In addition, 0.1 μg of pGMVβ plasmid DNA (Clontech, Palo Alto, CA, Cat. # 6177-1) was added as a control for transfection efficiency. After 5 hours, the culture medium was replaced with serum-free DMEM / BSA in the presence or absence of acetylated LDL (100 βg / ml) and incubated for 24 hours.

For the loaded convenience in high throughput screening, cultured cells can be stably transfected with a reporter plasmid using the following procedure. First, 5 × 10 ⁶ RAW264.7 cells in 10 ml serum-free DMEM containing 50 μl Geneporter transfection reagent (Gene Therapy Stratagene, San Diego, Calif.) Were treated with 9 μp pMPR1 plasmid and pCMVscript (Stratagene, CA). Transfect for 5 hours in a 60 mm dish. Subsequently, the transfection medium is replaced with complete medium and the cells are incubated overnight at 37 ° C. Subsequently, cells are transferred to separate dishes at dilutions ranging from 1: 5 to 1: 1000 and incubated for 20 days in selective medium containing 800 μg / ml G418 (Life Technologies, Bethesda, MD). Visible colonies were picked, grown and assayed for luciferase activity as described below. Using this method, five clonal cell lines positive for luciferase activity were identified for use in high throughput assays.

Luciferase assay : Following transfection, the cells in each well were treated with 70 μl of 1 × cell lysis reagent 8 Promega, Madison, WI, Cat. # E3971), subjected to one freeze-thaw cycle and the lysate was removed by centrifugation at 12,000 g for 5 minutes. After centrifugation, 100 μl of luciferase assay reagent (Promega, Madison, WI, Cat. # E1501) was added to 10 μl of lysate. The luciferase activity of each lysate was measured as light units using a luminometer. In addition, each lysate β-galactosylase using the chemiluminescent assay reagent supplied in the Galacto-light kit according to the manufacturer's instructions (Tropix Inc., Bedford, MA: Cat. # BL100G) Was measured. The normalized luciferase activity for each lysate is determined by dividing the luciferase activity value by the measured β-galactosylase value and reported as relative light units.

Results : The luciferase activity detected in cells transfected with pAPR1 was 3.3-fold higher than that detected in control cells transfected with pGL3-Basic plasmid containing only luciferase cDNA. These results indicated that the transcriptional regulatory region of ABC1 is in place. When cells transfected with pAPR1 were incubated with 100 μg / ml acetyl LDL for 24 hours, luciferase activity was 3.25 times higher than in cells not treated with acetyl LDL. These results suggest that the genomic ABC1 sequence contains a “cholesterol responsive” element found in the 5 ′ flanking region that mediates the cholesterol load response of the native ABC1 gene. This reporter system can also be used to test other compounds to determine whether a compound modulates ABC1 expression.

(Example 16)
This example shows a further assay that can be used to test compounds for their ability to modulate ABC1 gene expression using a reporter gene under the control of the endogenous ABC1 promoter.

  This assay involves constructing a recombinant vector containing a promoterless reporter gene and a selectable marker gene. The vector is linearized and transfected into the cell such that the reporter gene is integrated into the cell genome downstream of the endogenous ABC1 promoter. Using this assay, reporter gene expression is driven by the endogenous ABC1 promoter in response to the test compound.

Construction of a reporter plasmid : A recombinant vector containing a promoterless reporter gene can be made starting with a 7 kb EcoRI genomic fragment of ABC1 containing the exon 0 and intron 1 parts (including the ABC1 start site). . Site-directed mutagenesis can be used to generate a SalI restriction site in the exon 0 sequence upstream of two known start sites. The recombinant vector is generated by inserting a DNA fragment containing a promoterless reporter gene such as luciferase and a promoterless selectable marker such as puromycin resistance into the SalI site. An internal ribosome entry signal should be inserted between the reporter gene and the marker gene so that the gene is transcribed in the correct orientation. The recombinant vector contains two Eco47III sites, one of which must be eliminated using, for example, site-directed mutagenesis. The vector is linearized with the remaining Eco47III site located upstream of exon 0.

Transfection of reporter constructs : A linearized recombinant vector containing a reporter gene and a marker gene is introduced into cultured cells, including human cells, by any of a variety of transfection methods known in the art. For example, the linearized vector can be transfected using the method described in Example 15. A linearized recombinant vector contains ABC1 sequences that allow the vector to integrate into the cell genome at the site of the endogenous ABC1 gene. Addition of appropriate antibiotics to the culture medium allows selection of only those cells in which the reporter gene and marker gene are correctly oriented and integrated downstream of the endogenous ABC1 promoter. For example, if the vector contains a puromycin resistance gene inserted downstream of the reporter gene, the transfected cells should grow in the presence of puromycin. Only cells that have a suitably integrated puromycin resistance gene and are capable of encoding a functional protein will survive in the presence of puromycin. Thus, the transfected cells should grow under conditions that induce ABC1 promoter activity in the presence of the appropriate antibiotic. Surviving cells can be clonally cultured and DNA can be sequenced using PCR or Southern blot analysis and tested for proper integration of the genomic sequence.

  The resulting cells containing a reporter gene under the control of the endogenous ABC1 promoter can be used to determine whether a test compound modulates ABC1 expression. The ABC1 modulating activity of a compound is determined by assaying the level of reporter gene expression found in cells exposed to the test compound. For example, the cells having an integrated luciferase gene can be used to determine the ABC1 modulating activity of a test compound by measuring the amount of luciferase activity found in cells exposed to the compound.

(Example 17)
This example shows that ligands for nuclear receptors upregulate the expression of reporter genes under the control of the ABC1 promoter.

  In order to determine whether ligands for LXRα, LXRβ and RXRα nuclear reporters can regulate ABC1 gene expression, a pAPR1 plasmid containing a luciferase reporter gene under the control of the ABC1 promoter is transformed into at least one ligand for nuclear receptors. RAW264.7 cells that had been treated with <RTIgt; c </ RTI> were transfected (Fig. 12).

Reporter construct construction and transfection : Reporter construct pAPR1 and control reporter construct pGL3-Basic were obtained as described in Example 15. RAW264.7 cells were maintained in culture medium and transfected with either pGL3-Basic (1 μg) or pAPR (1 μg) as described in Example 15. Transfected RAW364.7 cells were treated with ethanol (EtOH) (0.1% v / v), 20 (S) -hydroxycholesterol (20 (S) OH-chol) (10 μM), 9-cis retinoic acid (9 Treatment with either cis (RA) (10αM) or both 20 (S) OA-chol and 9-cis RA (total 24 μM) Luciferase activity was measured and relative as described in Example 15. Reported as a unit of light.

Results : The results of this experiment are shown in FIG. Control cells transfected with pGL3-Basic showed no luciferase activity (data not shown). Compared to the EtOH control, cells transfected with pAPR1 produce a 19-fold increase in luciferase reporter activity in the presence of 20OH-col, a 16-fold increase in luciferase activity in the presence of 9-cis RA, and both Resulted in a 280-fold increase in luciferase activity in the presence of two ligands. These results indicate that both sterols and retinoids induce a strong transcriptional response from the ABC1 5 ′ flanking sequence in pAPR1. Furthermore, there is a clear synergistic effect of the two classes of compounds, as can be seen by the dramatic increase in luciferase activity found in cells treated with both ligands. LXRα and RXRα reporters are known to form active heterodimers. Thus, ligand-induced activation of both nuclear receptors can simultaneously result in an observed synergistic increase in transcription.

  These data indicate that hydroxysterols such as 20 (S) hydroxycholesterol, and retinoids such as 9-cis retinoic acid activate the ABC1 promoter, indicating that these and related compounds are ABC1 in macrophage cells. We show that it can be useful in the development of therapeutic compounds that increase expression and remove peripheral sites of excess cholesterol. In addition, the ABC1 promoter / reporter gene screening assay of the present invention can be used to screen for other compounds that increase ABC1 expression and to identify additional therapeutic compounds.

(Example 18)
This example shows further characterization of the ABC1 promoter region, including identification of LXR response elements.

  In order to determine which part of the 5 'flanking region of ABC1 retains transcriptional activity in response to nuclear ligand, various plasmids containing different portions of the 5' flanking region and the luciferase reporter gene were RAW264.7 cells treated with at least one ligand for the nuclear receptor were transfected. Using this system, a sterol response element corresponding to nucleotides 1480-1510 of SEQ ID NO: 3 was identified. The sterol response element contains the direct repeat-4 element TGACCGatagTAACCT. Confirmation of the sterol response element was obtained using site-specific mutagenesis and band-shift assay techniques.

Reporter construct construction : The reporter construct pAPR1 and the control reporter construct pGL3-Basic were obtained as described in Example 15. Moreover, a reporter construct containing any one of nucleotides 1-1523, 1080-1643, 1181-11643, 1292-1643 and 1394-1643 of SEQ ID NO: 3 was constructed. A reporter construct (GL-6a) containing nucleotides 1080-1623 of SEQ ID NO: 3 was constructed as described in Example 15. A reporter construct containing nucleotides 1-1532 of SEQ ID NO: 3 was constructed by digesting pAPR1 with SpeI and NheI and then religating the gel-purified vector fragment. A reporter construct containing nucleotides 1181 to 1643 first digests GL-6a with StyI , blunts the sticky ends with Klenow enzyme, digests the resulting vector with SacI , and then 462 base pair blunt- SacI sticky. It was constructed by isolating the terminal fragment. This was cloned into a vector obtained by digesting GL-6a with Acc65I , smoothing the sticky ends with Klenow enzyme, digesting with SacI , and then gel isolating the vector fragment. A reporter construct containing nucleotides 1292-1463 is obtained by serial digestion of GL-6a with Acc65I , blunting with Klenow enzyme, digestion with SacII , blunting with T4 polymerase, and then gel-single The release vector fragment was constructed by religation. A reporter construct containing nucleotides 1294-1643 digests GL-6a with Acc651 , blunted the ends with Klenow enzyme, subsequently digested with ApaI, blunted with T4 polymerase, and then gel-isolated. The vector fragment was constructed by religating.

Transfection of reporter constructs : RAW 264.7 cells are maintained in culture according to the method described in Example 15, and either pGL3-Basic (1 μg), pAPR1 (1 μg) or one of the other reporter constructs. I was transfected. Transfected RAW264.7 cells were treated with ethanol (EtOH) (0.1% v / v), 20 (S) -hydroxycholesterol (20 (S) OH call) (10 μM), 22 (R) -hydroxycholesterol ( 24 (either 22 (R) OH-chol) (10 μM), 9-cis retinoic acid (9-cis RA) (10 μM), or both 20 (S) OH-chol and 9-cis RA (20 μM total) Time processed. Luciferase activity was measured and reported as relative light units as described in Example 15.

Site-specific mutagenesis: The sterol response element corresponding to nucleotides 1480-1510 of SEQ ID NO : 3 was mutated into the 1080-1643 sequence using site-specific mutagenesis. Specifically, the response element containing the direct repeat-4 element TGACCGatagTAACCT was mutated to CTGCCATagTAGACCT using the GeneEditor system (Promega, Madison, WI) according to the manufacturer's protocol.

Gel-shift assay : Nuclear extracts were prepared from RAW264.7 cells by the method of Ohllsson et al., Cell, 45: 35-55 (1986). ³² P-labeled oligonucleotides (5 ng) corresponding to the LXR response element (TCGAGTGACCGATAGTAACCTCTCGA; SEQ ID NO: 56) and its mutated counterpart (TCGAGCTGCACATAGTAACCTCTCGA; SEQ ID NO: 57) were obtained from LXRα and LXRβ (Santa Cruz Biotech. No. SC-1591, Santa Cruz, Calif.) 20 μm HEPES in the presence or absence of antisera against RXR (Santa Cruz, Biotechnology, Cat. No. SC-774, Santa Cruz, Calif.), pH 7.9, 60 mM KCl, 1 mM MgCl ₂ , 1 mM DTT, 66.6 μg / ml poly ( DidC) and 10% glycerol individually incubated with 5 μg of nucleoprotein for 30 minutes at room temperature. The protein-DNA complex was applied to a 4% non-denaturing polyacrylamide gel for 1.5 hours at 150 V in 0.5 × TBE buffer. Protein-DNA complexes were detected by autoradiography of dried gels.

Results : Individual reporters containing 5 ′ flanking regions corresponding to nucleotides 1-1643 of SEQ ID NO: 3 (ie, pAPR1), 1-1532, 1080-1643, 1181-11643, 12921643, or 1394-1643) Transfection with the construct, each yielding the same result. All of the individual constructs produced a 3-4 fold increase in luciferase reporter activity in the presence of 20 (S) OH-chol or 22 (R) OH-chol compared to the EtOH control. Also, all of the individual constructs produced an 8 to 10-fold increase in luciferase reporter activity in the presence of 9-cis RA. In addition, transfection with either of the constructs resulted in oxysterol ligands (either (20 (S) OH-chol or 22 (R) OH-chol)) and retinoid ligands (9- In the presence of cis RA), a 25-50 fold increase in luciferase activity occurs, indicating a synergistic interaction. Each of the described constructs showed comparable levels of luciferase activity in response to the tested ligand, indicating that the shorter 5 'flanking sequence contained transcriptional regulatory sequences for sterols and retinoids . Specifically, these results indicated that the transcriptional regulatory sequences for sterols and retinoids are located in the 5 ′ flanking region corresponding to nucleotides 1394-1532 of SEQ ID NO: 3.

  These results show that a reporter construct containing a wild type sequence corresponding to nucleotides 1080-1643 of SEQ ID NO: 3 and a reporter construct containing a mutated sequence corresponding to nucleotides 1080-1643 of SEQ ID NO: 3. Confirmed by the luciferase activity used, where the sterol response element found at nucleotides 1480-1500 was mutated as described above. Transfection with the wild-type sequence produced a transcriptional response as measured by an increase in luciferase activity in the presence of either 20 (S) OH-chol or 9-cis RA alone, both present together with the ligand. A synergistic response was generated below. In contrast, transfection with the mutated sequence did not produce a transcriptional response in the presence of 20 (S) OH-chol or 22 (R) OH-chol. Transfection of the mutated sequence retained a reduced response to 9-cis RA, resulting in a 4-5 fold increase in transcriptional activity, rather than the 8-10 fold increase observed with the wild type sequence. Also, transfection of the mutated sequence abolished the synergistic transcription response observed in the presence of 20 (S) OH-chol and 9-cis RA together. These results were further confirmed by gel shift assay using a sterol consensus sequence (nucleotides 1480-1510) and its mutated counterparts. Gel-shift assay showed that the nuclear binding protein isolated from RAW264.7 cells bound to the sterol consensus sequence, but the nuclear protein did not bind to the mutated sequence. Furthermore, incubation of the nucleoprotein with a wild type sterol consensus sequence in the presence of LXR antiserum results in the formation of a supershifted complex (ie, antibody-protein-DNA complex) that binds to the nuclear receptor LXR. A sequence as a sterol response element was identified. In contrast, incubation of the nucleoprotein with the wild type sterol response element in the presence of RXR antiserum did not form a supershifted complex, indicating that RXR does not bind to this sequence. These results indicate that mutations that disrupt nucleoprotein binding to the consensus sequence abolish the transcriptional response to LXR ligand and decrease the response to RXR ligand. Furthermore, nuclear binding experiments performed in the presence of LXR or RXR antiserum confirmed that the consensus sequence found at nucleotides 1480-1510 is an LXR response element. This element also mediates a partial response to 9-cis RA.

  All references cited in this specification, including patents and publications, are hereby incorporated by reference in their entirety. While this invention has been described with emphasis on preferred embodiments of this invention, it is intended that variations of the preferred embodiments can be used and that the invention can be practiced otherwise than as specifically described herein. It will be apparent to those skilled in the art. Accordingly, this invention includes all modifications encompassed within the spirit and scope of the invention as defined by the appended claims.

1A-D shows control cholesterol efflux and cholesterol efflux in the presence of HDL and apoA-I from normal fibroblasts (1A, C) and fibroblasts (1B, D) from patients with Tangier disease. It is a graph which shows a result. Unfilled circles represent cholesterol efflux from cells not exposed to HDL or Apo AI, filled circles represent cholesterol efflux from cells exposed to ADL, filled diamonds to Apo AI Represents exposed cholesterol efflux. FIG. 2 is a graphical representation of a gene expression microarray analysis showing a comparison of gene expression found in cells from Tangier patients (TD1) and that found in normal cells, so that a total of 58,800 humans The cDNA was hybridized with cDNA prepared from mRNA of cAMP-treated TD1 cell cDNA (labeled with Cy3 dye) and with cDNA prepared from mRNA of cAMP-treated normal cells (labeled with Cy5 dye). FIG. 3 is a schematic diagram showing a restriction map of the recombinant expression vector pCEPhABC1 containing the open reading frame of the human ABC1 gene. FIG. 4 shows a comparison between the published human ABC1 amino acid sequence (GenBank, accession number AJ012376) and the presently disclosed and claimed human ABC1 amino acid sequence (“CVT”) having 60 additional amino acids at the N-terminus. It is a schematic diagram of the gene structure of human ABC1 shown. FIG. 5 shows the inhibition that ABC1 transport inhibitors 4,4-diisothiocyanostilbene-2, 2′-disulfonic acid (DIDS) and sulfobromophthalein (BSP) have on apoA-I-mediated cholesterol efflux. FIG. 2 is a graphical representation showing the effect, where unfilled circles indicate apoA-I-mediated cholesterol in the presence of BSP and filled circles indicate apoA-I-mediated cholesterol in the presence of DIDS. FIG. 6 is a graph showing the inhibitory effect of antisense ABC1 oligonucleotide on apoA-I-mediated cholesterol efflux, apoA-I-mediated cholesterol efflux in cells incubated without antisense oligonucleotide, 30 μM β- FIG. 5 shows apo AI-mediated cholesterol efflux in cells exposed to globin antisense oligonucleotide and apo AI-mediated cholesterol efflux in cells exposed to 30 μM ABC1 antisense oligonucleotide. FIG. 7 shows stimulation of apoA-I-mediated cholesterol efflux caused by ABC1 gene overexpression using RAW264.7 mouse macrophage cells stably transfected with an expression plasmid for human ABC1 (pCEPhABC1) FIG. 4 is a graphical representation showing apoA-I-mediated cholesterol efflux in control parental cells (without pCEPhABC1) and apoA-I-mediated cholesterol efflux in clonal cells (L3, L5, L6) transfected with pCEPhABC1. FIG. 8 shows exposure to albumin (filled bar), exposure to 8-bR-cAMP (unfilled bar), exposure to cholesterol-load (shaded bar), or cholesterol Reverse transcription showing the level of ABC1 gene expression in normal cells and cells from Tangier disease patients (TD1 and TD2) either exposed to load and subsequently exposed to ApoA-I (hatched bars) It is a graph display of a polymerase chain reaction (RT-PCR). FIG. 9 shows ethanol (0.1% v / v), 9-cis retinoic acid (9-cis RA; 10 μM), 20 (S) hydroxycholesterol (20 (S) -OH; 10 μM), or 9-cis RA. And is a graphical representation of the results of RT-PCR analysis showing the level of ABC1 gene expression in RAW264.7 cells exposed to either 20 and 20 (S) -OH (10 μM each). FIG. 10 shows normal fibroblasts in the presence of no additive (control), 8-Br-cAMP (1 mM), cholesterol (30 μg / ml) or cholesterol and 8-Br-cAMP (30 μg / ml and 1 mM, respectively). The results of immunoprecipitation analysis showing the level of cell-surface ABC1 protein found in cells (NL1, 10A) and Tangier disease patients (TD1, 10B) are shown. FIG. 11 is a schematic diagram showing a restriction map of the recombinant expression vector pAPR1 containing the 5 ′ flanking region of the ABC1 gene located upstream of the open reading frame of the luciferase reporter gene. FIG. 12 shows that EtOH (control), 20 (S) —OH (10 μM), 9-cis RA (10 μM), or both 20 (S) -OH and 9-cis RA (each with pARP1 in the presence of 10 μM). Figure 2 is a graphical representation showing the level of luciferase reporter gene expression induced in infected RAW264.7 cells. FIG. 13 is a schematic diagram of the 5 'flanking region of the ABC1 gene.

Claims (1)

  A pharmaceutical composition for treating Tangier disease comprising 20 (S) hydroxycholesterol and 9-cis retinoic acid as ligands for a nuclear receptor that increases the expression of ABC1 from monocyte cells of a mammalian subject.

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